al., 1983; Reed et al., 1985). It is not ... cell activation and then to identify the factor(s) that are ...... (Gunter et al., 1989; Zipfel et al., 1989a; Irving et al., 1989).
THE JOURNAL OF BIOL.OCICAL CHEMISTRY 0 1990 by The American Society for Biochemistry
Vol. 265, No. 21, Issue of July 25, pp. 12671-12678, 1990 Printed in U.S. A.
and Molecular Biology, Inc.
cDNA Cloning and Characterization a Cloned T Helper Lymphocyte*
of Interleukin
2-induced Genes in (Received for publication,
Daniel From
E. Sabath$, the Department
Patricia of Pathology
L. PodolinQ,
Paul
and Laboratory
Medicine,
Clonal expansion of antigen-specific lymphocytes is an important aspect of the immune response. Interleukin 2 (IL2) is largely responsible for the amplification of antigen-specific T cells. In this study, the changes in gene expression accompanying interleukin 2 stimulation of T cells are examined, using a cloned T helper lymphocyte line as a model system. To isolate cDNA clones of IL2-induced genes, a cDNA library was screened by differential hybridization. Twenty-one different cDNA clones were isolated by this method, comprising six glycolytic enzymes, vimentin, a-tubulin, &actin, y-actin, ERp99, elongation factor 2, ribosomal phosphoprotein Pl, the DNA-binding protein dbpB/YB-1, as well as seven clones which do not correspond to any previously described sequences. These clones are used to study the time course of expression and the sensitivity to cycloheximide inhibition of IL2induced mRNAs. In addition, the tissue specificity of the unidentified mRNAs is examined, and two of these are shown to be expressed at high levels in normal mouse brain, with much lower or undetectable levels in the other tissues tested. These cDNA clones will be useful in future studies to determine the molecular basis of IL2-induced gene expression.
One of the central unresolved issues in cell biology is how a peptide hormone’s membrane signal is transmitted to the cell nucleus to alter gene expression and how this early gene expression induces subsequent phenotypic changes. In the past several years, considerable data regarding the mechanism of signal transduction in T lymphocyte activation has been generated. Antigen stimulation has been shown to cause increases in intracellular calcium, accompanied by phosphatidylinositol cleavage and activation of protein kinase C (for review see Weiss and Imboden, 1987), as well as the induction * This work was supported in part by National Institutes of Health Grant GM-36962. The BIONET computer resource was supported by NIH Grant P41RR01685. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by National Institutes of Health Training Grant 5T32-GM-07170. Current address: Dept. of Laboratory Medicine, SB10, University of Washington, Seattle, WA 98195. I Supported by National Institutes of Health Grant T32-CA0194013.
( Supported by National Institutes of Health Training Grant 5T32-GM-07170. 11To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, University of Pennsylvania, 547 Clinical Research Bldg., 422 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-6856.
G. Comberll, University
and Michael of Pennsylvania,
November 8, 1989)
B. Prystowskyll Philadelphia,
Pennsylvania
19104
of many mRNA species (Zipfel et al., 1989a, 1989b). ILZ1 stimulation, which follows antigen stimulation, is a less well characterized process, with no good evidence for the involvement of calcium (Mills et al., 1985) or protein kinase C (Mills et al., 1988; Valge et al., 1988). It is known that stimulation of the IL2 receptor by IL2 results in changes in protein phosphorylation (Gaulton and Eardley, 1986; Kohno et al., 1986a), but there are no known phosphorylation substrates that have been shown to directly cause early gene expression events, such as the increase in c-myc mRNA levels (Kelly et al., 1983; Reed et al., 1985). It is not known what effect, if any, these early events have on later gene expression and DNA synthesis. Many investigators have approached the problem of signal transduction by isolating genes expressed early after cell stimulation, in order to identify regulators of cell activation. An alternative approach to linking signal transduction with gene expression is to identify genes that are induced late after cell activation and then to identify the factor(s) that are responsible for the induction of these genes. This approach may be especially useful in the identification of factors responsible for the regulation of DNA synthesis, itself a “late” event. The identification of late-acting regulatory factors will allow the study of signal transduction in reverse. For example, a phosphorylation event induced by the interaction of IL2 with its receptor can be given functional significance by demonstrating that a regulatory factor is activated by phosphorylation. In a previous study, a set of proteins was identified whose rates of synthesis increased significantly shortly before the onset of DNA synthesis in cloned T helper lymphocytes (L2 cells) stimulated with recombinant IL2 (Sabath et al., 1986). While one of these proteins was subsequently shown to be proliferating cell nuclear antigen/cyclin (Moore et al., 1987), the remaining proteins have not yet been identified. In order to study the regulation of expression of genes expressed during late activation, it is first necessary to obtain cDNA clones of these genes, both to identify them and to determine the contributions of transcriptional and post-transcriptional mechanisms to the regulation their expression. In this report, the isolation and characterization of cDNA clones representing IL2-induced genes are described, and the implications for the regulation of gene expression during ILB-induced proliferation are discussed. ’ The abbreviations used are: IL2, interleukin 2; DMEM CM, Dulbecco’s modified Eagle’s medium supplemented with 0.24 mM asparagine; 0.136 pM folic acid, 1.5 mM glutamine, 0.55 mM arginine, 1 InM pyruvate, 10 mM MOPS, pH 7.2,50 pM 2-mercaptoethanol, 100 units/ml penicillin, 100 kg/ml streptomycin, and 10 fetal calf serum; SSC, sodium chloride/sodium citrate buffer; SDS, sodium dodecyl sulfate; MOPS, 3-(N-morpholino)propanesulfonic acid; TAE, Tris acetate electrophoresis buffer; TBE, Tris borate electrophoresis buffer.
12671
IL2-induced MATERIALS
AND
METHODS
The L2 Cell Line-The derivation and maintenance of the L2 cloned T cell line have been described elsewhere (Glasebrook and Fitch, 1980). Briefly, the cells were maintained with weekly restimulation bv combining 0.5 to 1 X lo5 L2 cells with 6 X lo6 irradiated allogeneic spleen cells and 10 units/ml of highly purified human IL2 produced in Escherichia coli (a gift of the Cetus Corporation, Emervville. CA, Rosenberg et al.. 1984. and Wane et al.. 1984). in a volume of 1 ml. DMEM CM (Duibecco’s modified Eagle’s medium supplemented with 0.24 mM asparagine, 0.136 pM folic acid, 1.5 mM glutamine, 0.55 mM arginine, 1 mM pyruvate, 10 mM MOPS, pH 7.2, 50 pM 2-mercaptoethanol, 100 units/ml penicillin, 100 ug/ml streptomycin, and iO% fetal calf serum) in 24-well tissue culture plates-was incubated at 37 “C with 5% CO.,. Alternativelv. lo6 L2 cells were cultured with 40 x lo6 spleen cellsin 10 ml of DMEM CM containing 20 units/ml recombinant IL2 in 60-mm petri dishes (Falcon 1007). At the end of the weekly cycle, the cells were in a resting state, and greater than 95% of the cells had G, DNA content (Sabath et al., 1986). For most experiments, cells were used 6-8 days after being stimulated with antigen and IL2. For IL2 stimulation, L2 cells were purified from accessory cells by Ficoll-Hypaque density gradient centrifugation (specific gravity 1.077) and were resuspended at lo@/ ml in 10 ml of DMEM CM in 60-mm petri dishes. Recombinant human IL2 was added at a concentration of 100 units/ml. Enzymes-Enzymes used in these experiments were from New England Biolabs,. Beverly, MA; Bethesda Research Laboratories; Boehrinaer Mannheim Biochemicals: and Promena Biotec. Madison. “ I
WI.
-
RNA Isolation-Total cellular RNA was isolated from L2 cells by lysis in guanidinium isothiocyanate and density gradient centrifugation over CsCl (Chirgwin et al., 1979; Glisin et al. 1974). Total cellular RNA was used for RNA blotting. Poly(A)+ RNA was isolated by oligo(dT) column chromatography (Maniatis et al., 1982). The eluted RNA was subjected to a second oligo(dT) chromatography step for use in cDNA synthesis. RNA was quantitated by absorbance at 260 nm. cDNA Lihrary Construction-First strand cDNA was synthesized in the presence of 4 mM sodium pyrophosphate (Gubler and Hoffman, 1983). Second strand cDNA was synthesized using RNase H, DNA polymerase I, and E. coli DNA ligase (Okayama and Berg, 1982). The ends were made blunt by incubating with T, DNA polymerase, the DNA was size-fractionated on Sepharose CL-4B (Pharmacia LKB Biotechnology Inc.), internal EcoRI sites were protected by methylation with E. coli methylase, and EcoRI linkers were added to the DNA. Excess linkers were removed by EcoRI digestion and CL4B chromatography, and the cDNA was ligated into XgtlO arms (Stratagene, San Diego, CA). The DNA was packaged in vitro using a commercial kit (Promega), and the phage were plated on E. coli strain HflA. Synthesis of cDNA Probes-For synthesis of cDNA probes, first strand cDNA was synthesized as usual, except that the concentration of dCTP was 150 pM and 250 &i [a-32P]dCTP (>3000 Ci/mmol, Amersham Corp.) was added. The RNA strand was hydrolyzed by incubating the reaction mixture at 70 “C for 20 min in the presence of 0.1 M NaOH. The reaction mixture was neutralized by the addition of 1 N HCl, and unincorporated nucleotides were removed by spun column chromatography (Maniatis et al., 1982) through Sephadex G50 (Pharmacia LKB Biotechnology Inc.). Screening of cDNA Library-Phage were plated at 20,000 plaques/ plate on 20 x 20-cm tissue culture plates (Nunc, Roskilde, Denmark). Plaques were lifted onto Genescreen Plus membranes (Du Pont-New England Nuclear). Two lifts were made per plate. Filters were hybridized to radiolabeled cDNA probes and washed as described by Anderson and Axe1 (Anderson and Axel, 1985). Secondary screening was similar, except that about 100 phage were plated per loo-mm petri dish, and 82-mm circles of ColonyScreen filters (Du Pont-New England Nuclear) were used for plaque lifts. RNA Blot Analysis-Total RNA was prepared and 10 fig was dissolved in sample buffer (50% deionized formamide, 6% formaldehyde, 20 mM MOPS, pH 7.0, 5 mM sodium acetate, 1 mM EDTA), heated for 15 min at 68 “C, and electrophoresed in a 1% agarose gel containing 6% formaldehyde, 20 mM MOPS, pH 7.0, 5 mM sodium acetate. 1 mM EDTA, and 200 na/ml ethidium bromide. Following electrophoresis the RNA was visuilized by UV illumination to verify that equal amounts of RNA were loaded per lane and that the RNA was intact. The RNA was transferred by capillary blotting to GenescreenPlus membrane in 10 x SSC (1 X SSC = 0.15 M NaCl,
Gene Expression 0.015 sodium citrate, pH 7.0), the membrane was baked in uacuo for 2 h at 80 “C, and was hybridized and washed as with the library screening, except that polyuridylic acid was omitted. Labeling of DNA Probes-DNA probes were labeled by the random primer method (Feinberg and Vogelstein, 1983, 1984). Labeled DNA was separated from unincorporated nucleotides by spun column chromatography through Sephadex G-50. Specific activity was generally 5-10 X lo8 cpm/pg. DNA Blotting and Cross Hybridization--XgtlO DNA was prepared by standard methods (Davis et al., 1986). DNA was capillary blotted onto Hvbond N nvlon membranes (Amersham Corn.) in 10 X SSC. The blots were dried at room temperature, the DNA’was UV-crosslinked to the membrane, and hybridization and washing was the same as that done with RNA blots. To determine which cDNA clones contained inserts derived from the same mRNA, the inserts of the various clones were isolated, radiolabeled, and hybridized to the blots prepared from the cDNA clones isolated. DNA Seouence Armlvsis-Sinele-stranded Ml3 DNA was sequenced with Sequenase iUnited Siates Biochemical, Cleveland, OH), a modified form of T, DNA polymerase (Tabor and Richardson, 1987), according to the method of the manufacturer. Reaction products were electrophoresed through 6% polyacrylamide, 7 M urea in 1.5 X TBE (1 X TBE is 89 mM Tris borate, pH 8.0.89 mM boric acid, 2 mM EDTA). Sequence analysis and database searches were accomnlished usine the IFIND and FASTA-MAIL uroerams at the BIONET Natioial Computer Resource for Molecular”Biology (Intelligenetics, Inc., Mountain View, CA; Pearson and Lipman, 1988; Roode et al., 1988). RESULTS
cDNA Library Construction and Screening-Since previous studies showed that there were several proteins expressed at high levels immediately before the onset of ILS-induced DNA synthesis (Sabath et al., 1986), the possibility existed that the genes for these proteins were regulated by common mechanisms, resulting in coordinate expression during cell growth. To explore this possibility, it was necessary to isolate nucleic acid probes for IL2-induced genes, both to examine expression at the RNA level, as well as to explore the genomic structure of these genes. A XgtlO cDNA library was created using poly(A)+ RNA from L2 cells which had been stimulated with IL2 for 20 h. From previous experiments, this time point was known to represent the period just prior to the beginning of DNA synthesis (Lee et al., 1986). Approximately 300,000 distinct clones were obtained. The library was screened by differential hybridization, using as probes radioactive first strand cDNA synthesized using poly(A)+ RNA from either unstimulated L2 cells or cells that had been stimulated with IL2 for 20 h. Duplicate plaque lifts were hybridized to each probe, and cDNA clones were selected which hybridized more strongly to the probe made from stimulated cell RNA than from unstimulated cell RNA. These clones were then picked, replated at low density for plaque purification, and were screened as before. A total of 40,000 plaque-forming units were screened in the primary screening, and 90 positive clones were isolated and confirmed as positive by secondary screening. Cross-hybridization Studies-Since the differential screening method selects for cDNA clones representing abundant mRNAs,
the
clones
isolated
were
expected
to
be found
fre-
quently in the cDNA library and especially among those clones selected as IL2-induced. DNA blotting was used to identify those clones which cross-hybridized and were presumably identical. X DNA from the 90 clones was digested with EcoRI, separated by agarose gel electrophoresis, and blotted onto nylon membranes. The cDNA inserts were isolated from these clones and used as probes to identify those clones containing the same cDNA insert. The results of this analysis are summarized in Table I. The 90 original clones
IL2-induced
TABLE
Summar\ (‘lone Glycolytic A3
Tunes
tound
12673
Gene Expression I
of IL.%Induced cDNA clones mRNA
Sne
cDNA
Sm
Identity
fib
b
53
1.3
1266
Glyceraldehyde-3-phosphate
4 3 3 1 1
2.9 1.8 1.7 1.6 1.6
1840 1240 1130
Pyruvate kinase Triosephosphate isomerase Non-neuronal enolase Lactate dehydrogenase Phosphoglycerate mutase-hke
7 1 2
1.7 1.6 1.9 :3.0
1810 2660 666 1730
2 1 1 1
2.9 2.8 0.5 1.5
1990 2350 630 1810
ERp99 Ribosomal dbpH/YB-1
1 1 1 1 1 1 1
0.9
730 1320 1350 473 1280 ‘i60 2990
7 ?-contains 7 7 ‘7 ? 7
enzymes:
dehydrogenase Al-l.1 All.” A162 El”.2 F10.2 Cytoskeletal B4 B14 B27 El1 1
1125
proteins:
3
Other: B25 F18 B6.1 B22.2 Unidentified B8 B20 E4.2 ET.2 E11.2 F3 F.5
1580
clones:
2. 7 1.2 1.6 1.0 4.0
were found to consist of 21 distinct cDNAs. Clone A3 was the most commonly occurring ILP-induced sequence, as it was found in 53 of 90, or 59%, of the clones obtained after secondary screening. The remaining clones were found less frequently, with B4 occurring seven times, and Al4.1 four times. Three clones were isolated three times out of 90 each, two clones were found twice, and the remaining 13 clones were found once. The 21 unique cDNA clones were partially sequenced and were used to examine the time course of RNA accumulation, possible mechanisms for the accumulation, and tissue specificity of IL2-induced mRNAs. Prelimirxary Characterization of ILP-induced Clones-In order to verify that the cDNA clones selected represented IL2induced mRNAs and to determine the size of their messages, the cDNA inserts were isolated and used as probes for RNA blots. The results of these experiments are shown in Fig. 1 and are summarized in Table II. The steady-state levels of RNA for each cDNA clone were compared at 0 and 20 h after IL2 stimulation by RNA blot analysis. Of the clones isolated from the secondary screening, all had higher mRNA levels in IL2-stimulated cells, with increases from 1.2. to 13.6.fold over the levels in unstimulated cells (Table II). One clone, B20, did not hybridize to a distinct band, but rather to a large number of heterogeneous mRNAs, resulting in a smear (Fig. 1). It should be noted that in the L2 cell system, base-line levels of mRNA for a given gene can vary 5- to lo-fold, depending on the degree of quiescence of the cells at the beginning of the experiment. This results in different degrees of induction for the various mRNAs from experiment to experiment. For example, in separate experiments, the level of proliferating cell nuclear antigen/cyclin mRNA increased 5- to 40-fold following IL2 stimulation.” Similarly, whereas some of the increases in mRNA levels presented in Table II ’ P. M. Shipman-Appasamy, observations.
and
M. B. Prystowsky,
unpublished
Vimentin
c?-Tubulin /CA&n y-Actin
Elongation
factor 2 phosphoprotem
Pl
Bl repeat
FIG. 1. RNA blot analysis of ILZ-induced mRNA. Total RNA was isolated from L2 cells that were either unstimulated f-1 or stimulated with IL2 for 20 h (+l. 10 pg of RNA were separated by formaldehyde agarose gel electrophoresis, the RNA was transferred to nylon membranes by capillary blotting, and radiolabeled cDNA clones were hybridized to the blots. Equal RNA loading was verified by visualizing ethidium bromide-stained ribosomal RNA bands. Exposure times are different for each probe, depending on the abundance of each RNA species in the cells. The sizes of the various messages are estimated based on their relative sizes to the ribosomal RNA bands. kb, kilobase(
are minimal, larger increases have been observed in other experiments. Identification of cDNA Clones-The cDNA clones were subcloned into M13mp18 for sequencing. Partial sequences of at least 280 base pairs for each clone, in most cases from both ends, were obtained and compared with known DNA sequences in the NIH and EMBL databases. In this way, a number of clones were identified (Table I). The most abundant ILS-induced clone, A3, was found to be identical to
12674
ILB-induced
Gene Expression TABLE
II
Characteristics GADPH, glyceraldehyde-3-phosphate LDH, lactate dehydrogenase PGM,
triosephosphate 2.
Degree
Clone (mRNA)
A3 (GAPDH) A14.1 (PK) A14.2 (TPI) A16.2 (enolase) E12.2 (LDH) F10.2 (PGM-like) B4 (vimentin) B14 (a-tubulin) B27 (@-actin) Ell.l (y-actin) B25 (EF-2) F18 (ERp99) B6.1 (phosphoprotein B22.2 (dbpB/YB-1) B8 B20 E4.2
of expression of IL2-induced mRNAs dehydrogenase; PK, pyruvate kinase; TPI, phosphoglycerate mutase; EF-2, elongation factor of induction”
Pl)
E7.2 E11.2 F3 F5 a Fold increase in steady-state b Determined bv examinina ’ Not tested. ” d Not determined.
2.4 4.0 13.6 7.7 3.2 5.5 5.2 3.8 2.3 2.3 2.5 2.6 2.1 2.8
inhibition?
4.4
No No Yes No No Yes No No No No No No No No No NT No
2.3 6.9 2.8 2.1
No No No No
Tissue
b
isomerase;
specificity
NT
ET NT
ET NT ii: All tissues but pancreas Found in all tissues tested Found in all tissues tested Brain > heart, kidney, adrenal, colon, lung, skin, thymus Found in all tissues tested All tissues but pancreas Found in all tissues tested Brain > thymus > colon
RNA level at 20 h after IL2 stimulation (see Fig. 1). RNA levels 8 and 30 h after treating L2 cells with IL2 and 300 nm cycloheximide.
glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). Further sequence analysis showed that a number of other glycolytic enzyme cDNAs had been isolated by differential hybridization. Clone Al4.1, which was isolated four times was identified as pyruvate kinase (EC 2.7.1.40). Clone A14.2, isolated three times, was identified as triose-phosphate isomerase (EC 5.3.1.1), A16.2, also isolated three times, was nonneuronal enolase (EC 4.2.1.11), clone E12.2 was found to be lactate dehydrogenase (EC 1.1.1.27), and clone F10.2 was similar to phosphoglycerate mutase (EC 5.4.2.1). It should be noted that only part of clone F10.2 corresponded to human phosphoglycerate mutase, and the mRNA detected was 1.6 kilobases in size, different from the 0.8kilobase size reported for human phosphoglycerate mutase (Shanske et al., 1987), so this clone will be referred to as “phosphoglycerate mutaselike.” In addition to the glycolytic enzymes, the next most frequently occurring IL2-induced cDNAs encoded cytoskeletal proteins. The second most commonly occurring clone, B4, was shown to be vimentin, which was isolated seven times out of the original 90. Clone B27, isolated three times, was found to be @-actin, clone Ell.l, isolated twice, was found to be y-actin, and clone B14, which was isolated once, was found to be the cDNA for a-tubulin. Four other cDNAs have been identified based on their similarities to known sequences in the databases. Clone B25, isolated twice, was identified as elongation factor 2 (Kohno et al., 1986b). Clone F18, isolated once, was found to be ERp99, a 99-kDa transmembrane endoplasmic reticulum glycoprotein that shares some homology with the yeast heat shock protein, hsp90 (Mazzarella and Green, 1987). Clone B6.1 was identified as ribosomal phosphoprotein Pl (Rich and Steitz, 1987). Finally, clone B22.2 was most similar to a recently described DNA binding protein known as dbpB, which binds the epidermal growth factor receptor enhancer (Sakura et al., 1988),
or YB-1, which binds the Y box of the class II major histocompatibility antigen promoter (Didier et al., 1988). Clone B20 was found to contain a Bl repetitive sequence in its 3’ end, which resulted in considerable similarity to many sequences in the NIH database. When the 5’ end was compared with the sequences in the databases; however, no matches were found. Because of the Bl repeat, clone B20 hybridizes to many RNAs when used as a probe on RNA blots (Fig. l), resulting in a smear. Because it cannot be used for the analysis of a single RNA species, no further characterization was carried out on clone B20. Sequences from both ends of the remaining cDNA clones failed to match any sequences in the NIH or EMBL databases and therefore represent novel genes, all of which are ILP-inducible in L2 cells. Having characterized the sequences isolated, their expression following IL2 stimulation of L2 cells was examined. Expression of ILB-induced mRNA-Having established by differential hybridization and RNA blot analysis that the clones isolated did in fact represent IL2-induced genes, their expression was examined in greater detail. L2 cells were stimulated with IL2, and total RNA was isolated at 0, 8, 24, 30, and 48 h after stimulation. In addition, to determine whether protein synthesis was necessary for the induction of these mRNAs, the cells were stimulated with IL2 in the presence of 300 nM cycloheximide. This dose inhibits protein synthesis by 66%, based on [35S]methionine incorporation, completely inhibits IL2-induced DNA synthesis in L2 cells, as determined using flow cytometry to measure DNA content, but is not toxic to the cells for at least 48 h, as judged by the cells’ ability to exclude trypan blue (data not shown). Similar degrees of inhibition of protein synthesis and proliferation were observed by Pardee’s group, where the effect of protein synthesis inhibitors on serum-induced fibroblast proliferation was studied (Campisi et al., 1982). The cycloheximide was added at the time of IL2 stimulation, and the effect of cyclo-
IL2-induced
Gene Expression
heximide on RNA levels was examined at 8 and 30 h after IL2 stimulation. The results of these experiments are shown in Fig. 2. The first finding of note was that cycloheximide did not affect the level of expression of 17 of 20 ILS-induced mRNAs. In addition, cycloheximide-induced superinduction was not observed. This was somewhat surprising given that some of the mRNAs were maximally induced relatively late after IL2 stimulation. For three of the clones, cycloheximide caused partial inhibition of mRNA induction. ILB-induced TPI and phosphoglycerate mutase-like mRNA accumulation was inhibited by cycloheximide at 8 and 30 h after IL2 stimulation, and the induction of glyceraldehyde-3-phosphate dehydrogenase mRNA was partially inhibited at 8 h. When time course of induction was examined, a number of different patterns of expression were observed for the IL2induced genes. Most of the mRNAs examined were at maximal levels 8 h after IL2 stimulation. Another set of genes, including PK, cu-tubulin, ERp99, and the unidentified genes E11.2 and F5, had large increases between 8 and 24 h after IL2 stimulation. Taking the cycloheximide and time course data together, two conclusions can be drawn. First, whereas it might be expected that genes for the glycolytic enzymes would be coordinately expressed, it is apparent that there are a number of patterns of expression among them. Second, IL2stimulated proliferation is a complex process. IL2 stimulation Glycolylic H,lO CHX-
Enzymes
Cyloskeleial
I B 24 30 Jo 48 - + - - + mm....
Proteins
tlrs 0 8 8 1.3(1304* CHX - - + - - + GAPDH
@qqqa
12675
of the cells used in this study activates gene expression, which have different quirements for protein synthesis.
multiple pathways of time courses and re-
Tissue Specificity of the Unidentified Clones-Despite their being isolated from a T lymphocyte, most of the sequences isolated by differential hybridization are likely to be expressed in all tissues, given the basic cell functions they perform. On the other hand, there is no reason to assume that the unidentified clones and/or B22.2 (dbpB/YB-1) are similarly ubiquitous. In order to determine whether any of the unidentified genes were specific to murine lymphocytes, RNA was isolated from various normal mouse tissues, and RNA blots were prepared. The unknown cDNA clones were used as probes to determine their tissue specificity of expression. In addition, B22.2 (dbpB/YB-1) was used as a probe in this experiment, since as a DNA-binding protein, it was of interest to know whether its expression was restricted to certain tissues. Two of the six unidentified cDNA clones hybridized to mRNA that was expressed in every tissue examined and another two, as well as B22.2, to mRNA expressed in all tissues but pancreas (Fig. 3). However, E4.2 and F5 had an more restricted tissue distribution in which the highest levels of RNA were detected
Unidentified Ii,* 0 8 a 1. Y) 30.8 CHX- - + - - + -
“ime”ll”
* “, *. / BI
822.2
E4.2
E7.2
El 1.2
F3
FIG. 2. Time course of expression and cycloheximide sensitivity of IL2-induced mRNA. L2 cells were stimulated with IL2 for the indicated times either in the presence or absence of 300 nM cycloheximide. Total RNA was isolated at the times indicated and 10 pg were separated per lane. Equal loading was assessed by ethidium bromide staining of ribosomal RNA bands. A total of 10 identical blots were made, most of which were hybridized repeatedly. Exposure times vary from 2 to 72 h, depending on the abundance of the RNA examined. Note that, despite the presence of 300 nM cycloheximide, which completely inhibits ILZ-induced DNA synthesis, the accumulation of most RNAs was not inhibited, the exceptions being triosephosphate isomerase and the phosphoglycerate mutase-like mRNA, and to a lesser extent, glyceraldehyde-3-phosphate dehydrogenase.
F5
FIG. 3. Tissue specificity of unidentified IL2-induced mRNAs, as well as B22.2 (dbpB/YB-1). Total RNA was isolated from the indicated mouse tissues. and 10 pg was separated per lane on a 1% agarose/formaldehyde gel. Equal loading was assessed by ethidium bromide staining of ribosomal RNA bands. The indicated cDNA clones were hybridized to assess their tissue specificity. The autoradiographs were intentionally overexposed to detect minimal amounts of RNA in tissues where there was low abundance.
ILB-induced
12676
Gene Expression
in brain. E4.2 was also detectable at low levels in several other tissues, including heart, kidney, colon, lung, skin, and thymus. F5 RNA was present in modest amounts in the thymus, was barely detectable in the colon, and was not detected in any other tissue examined. DISCUSSION
Differential hybridization and/or subtractive hybridization has been used to study activation-inducible genes in both lymphoid and non-lymphoid systems, often with the goal of isolating genes important in the regulation of gene expression during cell proliferation or differentiation (St. John and Davis, 1979; Williams and Lloyd, 1979; Cochran et al., 1983; Linzer and Nathans, 1983; Hirschhorn et al., 1984; Lau and Nathans, 1985; Muscat et al., 1985; Burd et al., 1987; Kulesh et al., 1987; Leonard et al., 1987, Milbrandt, 1987; Schmid and Weissman, 1987; Almendral et al., 1988; Zipfel et al., 1989a). Most of these studies have concentrated on “immediate early” genes, those that are induced rapidly upon cell stimulation, often using cycloheximide to inhibit protein synthesis (Lau and Nathans, 1987; Almendral et al., 1988; Zipfel et al., 1989a), selecting for the very first genes expressed following a growth stimulus. Examples of serum-induced regulatory factors that are up-regulated early after stimulation include c-fos (Milbrand& 1987, Almendral et al., 1988), c-myc (Almendral et al., 1988), AP-l/c-jun (Ryder et al., 1988), egr-1 (Milbrandt, 1987; Sukhatame et al., 1988; Christy et al., 1988; Lemaire et al., 1988), as well as novel zinc finger-containing sequences (Almendral et al., 1988), all of which are known or suspected regulators of gene expression. Similar experiments have been carried out using the Jurkat T cell tumor line stimulated with phytohemagglutinin and phorbol ester (Zipfel et al., 1989a). Although no known regulators of gene expression were isolated in these experiments, two novel cDNAs were characterized having sequences suggesting that they may be cytokines (Zipfel et al., 1989b). These would presumably mediate cellcell communication during the immune response and therefore would regulate cell growth or differentiation genes indirectly. Although the isolation of immediate early cDNAs has yielded a number of genes that are likely to regulate genes induced later in the growth cascade, it is not known which genes are the targets of these factors. For this reason, we elected to isolate genes expressed maximally during late activation. By studying the regulation of expression of these genes, we would be able to determine which factors are important in the induction of proliferation-associated genes. Although the majority of the IL2-induced cDNAs isolated can be considered “housekeeping” genes, a potential regulatory gene has been isolated, dbpB/YB-1. This gene has the property of being induced by IL2, but the protein product may regulate the expression of other genes in turn. It was initially characterized by its ability to bind specifically to both the Y box of the major histocompatibility complex class II promoter (Didier et al., 1988) and the enhancer of the epiderma1 growth factor receptor (Sakura et al., 1988), both of which contain inverted CAAT elements (Didier et al., 1988; Maekawa et al., 1989). Interestingly, a number of growth-associated genes also contain inverted CAAT sequences in their promoters, including thymidine kinase (Lipson et al., 1989), proliferating cell nuclear antigen/cyclin (Travali et al., 1989), and DNA polymerase (Y(Pearson et al., 1989). In addition the promoters of these genes all lack TATA boxes. It is tempting to speculate that these genes are all coordinately regulated during cell proliferation and that dbpB/YB-1 is involved in
growth-associated gene expression, including ILS-induced T cell proliferation. With regard to mechanisms involved in the induction of activation-associated genes, it is apparent that there is considerable complexity in the activation cascade, as evidenced by the different patterns of expression that are observed among ILP-induced genes. Among the 21 genes examined in this paper, we observe differences in degree of mRNA induction, time course of induction, the effect of cycloheximide on induction, as well as the tissue distribution of expression. This type of heterogeneity has been observed in serum-stimulated fibroblasts (Linzer and Nathans, 1983; Almendral et al., 1988) and lectin/phorbol ester-stimulated lymphocytes (Gunter et al., 1989; Zipfel et al., 1989a; Irving et al., 1989). These data imply the existence of multiple divergent pathways that are set into motion by the interaction of IL2 with its receptor. With three exceptions, the ILP-induced mRNAs accumulate in the presence of concentrations of cycloheximide which completely inhibit DNA synthesis and inhibit protein synthesis by 66%. The phenomenon of superinduction is not observed for any of these mRNAs, in contrast with many mitogen-induced immediate early genes studied by other investigators (Lau and Nathans, 1987; Almendral et al., 1988; Zipfel et al., 1989a). Three conclusions can be drawn from these data. First, the induction of most of the genes in this study does not depend on the synthesis of immediate early proteins, but rather depends on an alteration in activity of proteins already present. This alteration could be caused by a posttranslational modification such as phosphorylation or by alteration in activity due to the accumulation of some substance in the cells or culture medium. Second, the three mRNAs whose accumulation is inhibited by cycloheximide are presumably regulated by different mechanisms than the others. Finally, the factors regulating the onset of IL2-induced DNA synthesis are distinct from those regulating ILB-induced RNA accumulation. Because of their delayed appearance, we might have expected that these mRNAs would have their induction inhibited under conditions where DNA synthesis, a concurrent event, was inhibited. Presumably, the commitment to begin DNA synthesis occurs after the onset of expression of most of the mRNAs examined in this study, suggesting that a “restriction point,” as described by Pardee (1974), exists for IL2-driven activation. Although we have no data addressing the molecular mechanisms involved in the induction of these activation-associated genes, there is one clone that has a sequence element which could be responsible for its induction by IL2. Clone B20, which was not identified but contains a Bl repetitive element, was found to hybridize to a large number of mRNAs on RNA blots, resulting in a smear. A similar finding was reported in the rat, where serum-induced cDNA clones were isolated that contained the repetitive identifier (ID) sequence, and hybridized to heterogeneous RNAs (Glaichenhaus and Cuzin, 1987). In that study, the ID element was able to confer serum-inducibility when linked to a /3-globin gene, and a posttranscriptional mechanism was responsible for the induction. The murine equivalent of the ID element is the Bl repeat.3 Clone B20, therefore, probably represents a family of activation-associated genes that are induced by IL2, although it remains to be shown whether the Bl repetitive element can confer IL2 inducibility upon a reporter gene in T lymphocytes. In addition to the feature of being induced during IL2stimulated L2 cell proliferation, two of the clones, E4.2 and F5, appear to be regulated in a tissue-specific pattern. The ” N. Glaichenhaus,
personal
communication.
IL2-induced
fact that E4.2 and F5 are expressed at high levels in brain is of interest given the features common to the nervous system and the immune system. Both T lymphocytes and neural tissue express Thy-l in the mouse, and lymphocytes express neuropeptides such as preproenkephalin (Kwon et al., 1987; Zurawski et al., 1986) and endorphinand adrenocorticotropic hormone-like peptides (Harbour-McMenamin, 1985, Russell et al., 1985). In addition, they bear prolactin receptors (Russell et al., 1985), and lymphocyte function can be modulated by adrenocorticotropic hormone (Johnson et al., 1982, 1984), methionine-enkephalin (Wybran et al., 1979), and p-endorphin (Gilman et al., 1982). Although the physiologic significance of these findings is not yet clear, they do suggest some common link between the immune system and the nervous system. We would speculate that F5 and E4.2 represent other gene products common to lymphocytes and neurons or glial cells. T lymphocyte activation can be thought of as occurring in two stages. In the first stage, the interaction of the antigen receptor with its cognate antigen results in the transition from Go to G1, the expression of early activation mRNAs, and the production of lymphokines, including IL2 and the IL2 receptor. The second stage is initiated by the interaction of IL2 with its receptor, driving the T cells into S phase, expanding the population of antigen-stimulated cells. In this study we have isolated genes that are more closely associated with this second phase of activation, using ILZ-stimulated cloned T cells as a model system. By combining the results of the early activation experiments (Zipfel et al., 1989a) with ours, a fairly complete picture of the genomic response of T cells to antigen stimulation can be assembled, from a resting state through G1 activation and DNA synthesis. It will be of interest to see whether any of the early genes directly influence the expression of later IL2-induced genes. Now that some genes have been identified that are expressed in various stages of T cell activation and proliferation, it is possible to begin addressing the mechanisms responsible for these processes. Acknowledgments-We would like to thank Dr. Mitchell J. Weiss who helped us make the cDNA library. We also thank Nancy Thornton for technical assistance. REFERENCES Almendral, J. M., Sommer, D., MacDonald-Bravo, H., Burckhardt, J., Perera, J., and Bravo, R. (1988) Mol. Cell. Biol. 8, 2140-2148 Anderson, D. J., and Axel, R. (1985) Cell 42, 649-662 Burd, P. R., Freeman, G. J., Wilson, S. D., Berman, M., DeKruyff, R., Billings, P. R., and Dorf, M. E. (1987) J. Zmmunol. 139, 31263131 Campisi, J., Medrano, E. E., Morreo, G., and Pardee, A. B. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 436-440 Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299 Christy, B. A., Lau, L. F., and Nathans, D. (1988) Proc. N&l. Acad. Sci. U. S. A. 85, 7857-7861 Cochran, B. H., Reffel, A. C., and Stiles, C. D. (1983) Cell 33, 939947 Davis, L. G., Dibner, M. D., and Battey, J. F. (1986) Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., New York Didier, D. K., Schiffenbauer, J., Woulfe, S. L., Zacheis, M., and Schwartz, B. D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 73227326 Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13 Feinberg, A. P., and Vogelstein, B. (1984) Anal. Biochem. 13’7, 266267 Gaulton, G. N., and Eardley, D. D. (1986) J. Zmmunol. 136, 24702477 Gilman, S. C., Schwartz, J. M., Milner, R. J., Bloom, F. E., and Feldman, J. D. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 42264230
12677
Gene Expression Glaichenhaus, Glasebrook,
N., and Cuzin, A. L., and Fitch,
F. (1987) Cell 50, 1081-1089 F. W. (1980) J. Exp. Med.
151, 876-
895 Glisin, V., Crkvenjakow, R., and Byus, C. (1974) Biochemistry 13, 2633-2637 Gubler, U., and Hoffman, B. J. (1983) Gene (Amst.) 25, 263-269 Gunter. K. C., Irving, S. G., Zipfel. P. F., Siebenlist, U., and Kelly, K. (1989) J.‘Zmmunol. 142,3286-3291 Harbour-McMenamin. D.. Smith. E. M.. and Blalock. J. E. (1985) Infect. Zmmunol. 48; 813-817 ’ ’ Hirschhorn, R. R., Aller, P., Yuan, Z.-A., Gibson, C. W., and Baserga, R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 6004-6008 Irving, S. G.. June, C. H., Zipfel, P. F., Siebenlist, U., and Kelly, K. (1989) Mol. Cell. Biol. 9, 1034-1040 Johnson. H. M.. Smith. E. M.. Torres. B. A.. and Blalock. J. E. (1982) Proc. katl. Acad. Sci U. S.‘A. 79, 4171-4174 ’ Johnson, H. M., Torres, B. A., Smith, E. M., Dion, L. D., and Blalock, J. E. (1984) J. Zmmunol. 132,246-250 Kelly, K., Cochran, B. H., Stiles, C. D., and Leder, P. (1983) Cell 35, 603-610 Kohno, M., Kuwata, S., Namba, Y., and Hanaoka, M. (1986a) FEBS Lett. 198.33-37 Kohno, K., Uchida, T., Ohkubo, H., Nakanishi, S., Nakanishi, T., Fukui. T.. Ohtsuka. E.. Ikehara. M.. and Okada. Y. (1986b) Proc. Natl. kcad. Sci. U. S. A. 83, 4978-4982 Kulesh, D. A., Clive, D. R., Zarlenga, D. S., and Greene, J. J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8453-8457 Kwon, B. S., Kim, G. S., Prystowsky, M. B., Lancki, D. W., Sabath, D. E., Pan, J., and Weissman, S. M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,2896-2900 Lau, L. F., and Nathans, D. (1985) EMBO J. 4,3145-3151 Lau, L. F., and Nathans, D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1182-1186 Lee, S. C., Sabath, D. E., Deutsch, C., and Prystowsky, M. B. (1986) J. Cell Biol. 102, 1200-1208 Lemaire, P., Revelant, O., Bravo, R., and Charnay, P. (1988) Proc. Natl. Acad. Sci. U. S. A. 85.4691-4695 Leonard, D. G. B., Ziff, E. B:, and Greene, L. A. (1987) Mol. Cell. Biol. 7,3156-3167 Linzer, D. I. H., and Nathans, D. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,4271-4275 Lipson, K. E., Chen, S.-T., Koniecki, J., Ku, D.-H., and Baserga, R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6848-6852 Maekawa, T., Imamoto, F., Merlino, G. T., Pastan, I., and Ishii, S. (1989) J. Biol. Chem. 264, 5488-5494 Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Mazzarella, R. A., and Green, M. (1987) J. Biol. Chem. 262, 88758883 Milbrandt, J. (1987) Science 238, 797-799 Mills, G. B., Cheung, R. K., Grinstein, S., and Gelfand, E. W. (1985) J Zmmunol. 134,2431-2435 Mills, G. B., Girard, P., Grinstein, S., and Gelfand, E. W. (1988) Cell 55,91-100 Moore, K. S., Sullivan, K., Tan, E. M., and Prystowsky, M. B. (1987) J. Biol. Chem. 262,8447-8450 Muscat, G. E. O., Caputo, A., McCairns, E., and Rowe, P. B. (1985) DNA 4,377-384 Okayama, H., and Berg, P. (1982) Mol. Cell. Biol. 2, 161-170 Pardee, A. B. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 1286-1290 Pearson, B. E., Nashouer, H.-P., and Wang, T. S.-F. (1989) J. Cell. Biochem., Suppl. 13D, 169 Pearson, W. R., and Lipman, D. J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2444-2448 Reed, J. C., Sabath, D. E., Hoover, R. G., and Prystowsky, M. B. (1985) Mol. Cell. Biol. 5, 3361-3368 Rich, B. E., and Steitz, J. A. (1987) Mol. Cell. Biol. 7, 4065-4074 Roode, D., Liebschutz, R., Maulik, S., Friedemann, T., Benton, D., and Kristofferson, D. (1988) Nucleic Acids Res. 16, 1857-1859 Rosenberg, S. A., Grimm, E. A., McGrogan, M., Doyle, M., Kawasaki, E., Koths, K., Mark, D. F. (1984) Science 223, 1412-1415 Russell, D. H., Kibler, R., Matrisian, L., Larson, D. F., Poulos, B., and Magun, B. E. (1985) J. Zmmunol. 134,3027-3031 Ryder, K., Lau, L. F., and Nathans, D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,1487-1491 Sabath, D. E., Monos, D. M., Lee, S. C., Deutsch, C., and Prystowsky, M. B. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,4739-4743
IL2-induced Sakura, H., Maekewa, T., Imamoto, F., Yasuda, K., and Ishii, S. (1988) Gene (Am&.) 73, 499-507 Schmid, J., and Weissmann, C. (1987) J. Zmmunol. 139, 250-256 Shanske, S., Sakoda, S., Hermodson, M. A., DiMauro, S., and Schon, E. A. (1987) J. Biol. Chem. 262,14612-14617 St. John, T. P., and Davis, R. W. (1979) Cell 16, 443-452 Sukhatme, V. P., Cao, X., Chang, L. C., Tsai-Morris, C.-H., Stamenkovich, D., Ferreira, P. C. P., Cohen, D. R., Edwards, S. A., Shows, T. B., Curran, T., Le Beau, M. M., and Adamson, E. D. (1988) Cell 53,37-43 Tabor, S., and Richardson, C. C. (1987) Proc. Natl. Acad. Sci. U. S. A. 84.4767-4771 Travali, S., Ku, D.-H., R&o, M. G., Ottavio, L., Baserga, R., and
Gene Expression Calabretta, B. (1989) J. Biol. Chem. 264, 7466-7472 Valge, V. E., Wong, J. G. P., Datlof, B. M., Sinskey, A. J., and Rao, A. (1988) Cell 55, 101-112 Wang, A., Lu, S. D., and Mark, D. F. (1984) Science 224, 1431-1433 Weiss, A., and Imboden, J. B. (1987) Adu. Zmmunol. 41, l-38 Williams, J. G., and Lloyd, M. M. (1979) J. Mol. Biol. 129, 19-35 Wybran, J., Appelboom, T., Famaey, J.-P., and Govaerts, A. (1979) J. Zmmunol. 123,1068-1070 Zipfel, P. F., Irving, S. G., Kelly, K., and Siebenlist, U. (1989a) Mol. Cell. Biol. 9, 1041-1048 Zipfel, P. F., Balke, J., Irving, S. G., Kelly, K., and Siebenlist, U. (1989b) J. Immunol. 142, 1582-1590 Zurawski, G., Benedik, M., Kamb, B. J., Abrams, J. S., Zurawski, S. M., and Lee, F. D. (1986) Science 232,772-775
