Activation of lymphokine genes in T cells: Role of cis-acting DNA elements that respond to T cell activation signals

p/tarmac. Ther. VoI. 55, pp. 303-318, 1992 Printed in Great Britain. All rights reserved

0163-7258/92 $15.00 © 1993 Pergamon Press Ltd

Associate Editor: S. PESTKA

ACTIVATION OF LYMPHOKINE GENES IN T CELLS: ROLE OF CIS-ACTING D N A ELEMENTS THAT RESPOND TO T CELL ACTIVATION SIGNALS NAOKO ARAI,*¶ YOSHIYUKI NAITO,* MITSUO W A T A N A B E , * ESIV_aA~ S. MASUDA,* YUKO YAMAGUCHI-IWAI,*J~ AKXOTSUBOI,* Tosmo HEIKE,*§IKUO MATSUDA,* KYOKO YOKOTA,* NAOKO KOYANO-NAKAGAWA,'~ HYUN JUN LEE,~" MASAAKI MURAMATSU,~ TAKASHI YOKOTAJf a n d KEN-ICHI ARAIt

*DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA, U.S.A. *Department of Molecular and Developmental Biology, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan Abstract--Activation of T cells is initiated by the recognition of antigen on antigen presenting cells to exert the effector functions in immune and inflammatory responses. Two types of helper T cell (Th) clones (Thl and Th2) are defined on the basis of different patterns of cytokine (lymphokine) secretion. They determine the outcome of an antigenic response toward humoral or cell-mediated immunity. Although lymphokine genes are coordinately regulated upon antigen stimulation, they are regulated by the mechanisms common to all as well as those which are unique to each gene. For most lymphokine genes, a combination of phorbol esters (phorbol 12-myristate 13 acetate, PMA) and calcium ionophores (A23187) is required for their maximal induction. Yet phorbol ester alone or calcium ionophore alone produce several lymphokines.The production of the granulocyte-macrophage colony stimulating factor (GMCSF) is completely dependent on the two signals. We have previously found a cis-acting region spanning the GM-CSF promoter region (positions - 95 to + 27) that confers inducibility to reporter genes in transient transfection assays. Further analysis identified three elements required for efficient induction, referred to as GM2, GC-box and conserved lymphokine element (CLE0). GM2 defines a binding site for protein(s) whose binding is inducible by PMA. One protein, NF-GM2 is similar to the transcription factor NF-kB. GC-box is a binding site for constitutively bound proteins. CLE0 defines a binding site for protein(s) whose optimum binding is stimulated by PMA and A23187. Viral trans-activators such as Tax (human T cell leukemia virus-l, HTLV-1) and E2 (bovine papilloma virus, BPV) proteins are other agents which activate lymphokine gene expression by bypassing T cell receptor (TCR) mediated signaling. The trans-activation domain of E2 and Tax is interchangeable although they have no obvious sequence homology between them. The viral trans-activators appear to target specific DNA binding protein such as NF-kB and Spl to cis-acting DNA site and promote lymphokine gene expression without TCR-mediated stimulation.

CONTENTS 1. Introduction: The Cytokine Signal Network 2. T Cell Activation and the Regulation of Cytokine Genes 3. Differential Expression of Cytokines in Thl and Th2 Clones

304 305 306

~:Present address: The Institute for Virus Research, Kyoto University, Kyoto, Japan. §Present address: Department of Pediatrics, Kyoto University, Kyoto, Japan. ¶Corresponding author. Abbreviations---BPV, bovine papilloma virus; CLE, conserved lymphokine element; G-CSF, granulogyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony stimulating factor; HTLV, human T cell leukemia virus; IFN, interferon; IL, interleukin; LTR, long terminal repeat; PMA, phorbol 12-myristate 13 acetate; Th ceil, helper T cell; TCR, T cell receptor; TNF, tumor necrosis factor. n,r SS~--H

303

304

N. AgAI et al.

4. Regulation of the Expression of the GM-CSF Gene: Involvement of Oncogenes as Transcription Activators 4.1. GM2 defines an activation-dependent binding site for NF-GM2 4.2. The GC-box is a binding site for constitutive proteins 4.3. CLE0: a link between PMA and calcium signals? 5. Role of Viral trans-Activators in Lymphokine Gene Expression 6. Conclusion and Speculations References

309 309 310 311 311 315 316

1. INTRODUCTION: THE CYTOKINE SIGNAL NETWORK When healthy individuals are exposed to a foreign antigen, specific immune responses are stimulated to produce antibodies through a series of events involving multiple cellular interactions. In this cellular network, T cells, B cells and macrophages play a central role. These cells coordinate their activities through elaboration of cytokines. Proliferation and differentiation of mammalian cells such as epithelial, neuronal, lymphoid and hemopoietic cells are generally controlled by growth and differentiation factors. In the 1980s, following the identification of several growth factors such as epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF) and insulatin-like growth factor (IGF), molecular cloning of genes encoding cytokines produced by activated T cells and macrophages made remarkable progress. Although the hallmark of the immune system is specificity, i.e. the specific cellular recognition of foreign antigens by receptors on the surface of T cells and B cells, characterization of many cytokines revealed novel features of the immune system. Unlike immunoglobulins, the effects of cytokines are generally not antigen-specific. In cooperation with antigenic stimuli, cytokines amplify specific immune responses by supporting proliferation of a small number of lymphocytes specific for any one antigen (clonal expansion) and by recruiting multiple effector mechanisms required to eliminate foreign antigen. Multiple cytokines appear to control T cells and B cells positively or negatively through a cytokine network (for review see Arai et aL, 1990). IL-6o7. I I IFN-~, M-CSF G-CSF,GM-CSF

I 'zr::°/:::::'u°*r

/ TNF, G-CSF GM-CSF IFN-¢c

IL-3,4,5, 6~ IO, TNF GM-CSF

\ I=

~

9, I0, TNF,LT, GM-CSF,IFN-7

~

l, A, D,E, G,M

FIG. 1. The cytokine network and multiple cellular interactions in immune and inflammatory responses.

Lymphokine genes in T cells

305

While T cells are the major source of these factors (lymphokines), macrophages (monokines) and possibly B cells produce overlapping as well as distinct sets of factors (Fig. 1). The glycoproteins, such as interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, GM-CSF, granulocyte colony-stimulating factor (G-CSF), interferon-T (IFN-T), tumor necrosis factor • (TNF-~) and lyrnphotoxin mediate immune and inflammatory responses. Likewise, factors produced by stromal cells, such as macrophage-colony stimulating factor (M-CSF), IL-7, IL-11, leukemia inhibitory factor (LIF) and steel factor (c-kit ligand) also participate in these responses. Along with IFN-~, IFN-/~, tumor growth factor-~ (TGF-~) and TGF-/~, all these molecules are collectively called cytokines. Cytokines are defined as protein mediators for cell--cell communication involved in viral infection (IFNs), inflammation and immunity (monokines, lymphokines) and hemopoiesis (CSFs). Hemopoietic cells are produced continuously from self-renewing pluripotential hemopoietic stem cells in the bone marrow microenvironment, namely steady state (constitutive) hemopoiesis, which is distinguished from inducible hemopoiesis. Cytokines regulate both inducible hemopoiesis, that takes place following invasion of foreign antigen and steady state (constitutive) hemopoiesis, which operates independent of immunological stimulus. Cytokines were initially named after their biological activities on specific target cells, assuming that cytokines acted in a colinear fashion through a series of cell lineage-specific interactions and that no crosstalk between cells of different lineages were involved. However, such an assumption of cytokine action in the immune response is apparently far from a real picture. The lessons we have learned are the following. First, a single cytokine can interact with more than one type of cell. Second, a single cytokine has multiple biological activities. Third, a single cell can interact with more than one cytokine. And finally, many cytokines have overlapping activities. For example, cytokine receptors are expressed on multiple cell types and cytokines exert their functions through a cytokine network established between lymphoid cells, hemopoietic cells, endothelial cells and other target cells. In this cytokine network, a single cytokine can act both as a positive and a negative signal depending on the cell type of target cells. This network is controlled either at the inducer phase (i.e. the regulation of the expression of cytokine genes of producer cells) or at the effector phase (i.e. signal transduction via cytokine receptors on target cells).

2. T CELL ACTIVATION AND THE REGULATION OF CYTOKINE GENES Activation of T cells to exert their effector functions is initiated by the recognition of antigen on antigen presenting cells. The antigen signal received by the T cell receptor (TCR)/CD3 complex is transmitted to the nucleus, resulting in induction of a number of genes such as cytokines, immediate early response genes and oncogenes. A model for the TCR-mediated cascade of biochemical events is shown in Fig. 2, but much remains to be proven. The major events of early activation involve (1) plasma membrane inositol phospholipid hydrolysis, (2) increases in cytoplasmic calcium concentration, (3) phosphorylation of membrane and cytoplasmic proteins. Tyrosine kinases such as lck and fyn have received attention as molecules which may play an important role in coupling the TCR to signal transduction machinery downstream (Klausner and Samelson, 1991). Activation of p21 ~ following stimulation of the TCR suggests the involvement of the ras protooncogene in T cell activation (Downward et al., 1990). A role has been proposed for trimeric G protein in triggering phosphatidylinositol (PI) turnover through TCR-mediated signaling, however, evidence to support this notion is elusive (Imboden et al., 1986). The involvement of one or more isozymes of protein kinase C in TCR-mediated signaling has been established by experiments using phorbol ester or constitutively active protein kinase C (Muramatsu et al., 1989). Expression of cytokine genes appears to be regulated at least in part at the post-transcriptional level. The mRNAs of cytokines or the c-fos protooncogene, whose expression is transient, frequently contain an AUUA sequence in the Y-untranslated region (Shaw and Kamen, 1986). This sequence controls the stability of mRNAs in cis by interacting with a regulatory protein(s) whose half-life appears to be very short. The contribution of transcriptional or post-transcriptional mechanisms in the GM-CSF gene expression differs depending on cell types and on the method of stimulation (Razanajaona et al., 1992). Under certain conditions, antibodies against the TCR

306

N. ARAZet al.

L

Class MHCII

~

APC

S

CD45

T2~30~

~,.

)

Tyr'~,PTcR c

, :,r,,,n-'l~,

Tyr~ P phosphotose

IL-4

.... i

,,,

~

z

± _~/-I

Caz÷

y;

~

t,r o n s c r l p ~ e $

i

Ca2~

~

lyr ,~ p '

°"

FIG. 2. Scheme for T cell activation to produce lymphokines. or its associated CD3 molecule mimic the effect of antigen stimulus in T cell activation (Table 1). A combination of phorbol ester (PMA) and calcium ionophore (A23187) is required for maximal activation of cytokine genes, suggesting a synergy between the processes mediated by Ca ~+ and by protein kinase C. The production of IL-2 or GM-CSF shows such synergy between these two signals. Yet certain cytokines are activated by phorbol esters alone (IL-5, IL-6 and IL-10 ) or Ca 2+ ionophore alone (IL-3, IL-4 and IFN-~) (Table 1) indicating that, for some cytokines, one pathway dominates and that the balance between the activities of the two signaling pathways contributes to the patterns of cytokine expression. An immunosuppressive agent, cyclosporin A (CsA), almost completely inhibits production of those cytokines (IL-2, IL-3, IL-4, GM-CSF or IFN-7) whose expression depends on a calcium signal when T cells are activated by PMA/A23187 or antiCD3 antibody. In contrast, production of IL-5, IL-6 or TL-10 is only partially inhibited, regardless of the method of stimulation. To account for the coordinate regulation of a panel of lymphokines in T cells, one might assume a consensus DNA-sequence motif recognized by activation-specific DNA binding protein(s). Characterization of the GM-CSF and IL-3 genes revealed short stretches of homologous elements which we designated CLE1 (Conserved Lymphokine Element-l) and CLE2. These motifs are found in other lymphokine genes as well (Miyatake et al., 1985; Stanley et al., 1985). Recently, an additional element (CLE0) which shares some homology with the NF-AT sequence of the IL-2 enhancer has been found in the regulatory regions of TL-4, IL-5 and GM-CSF genes. The role of these elements in the regulation of lymphokine genes is described below.

3. DIFFERENTIAL EXPRESSION OF CYTOKINES IN TH1 AND TH2 CLONES In the course of cloning lymphokine cDNAs, we recognized that mouse T cell clones secrete a distinct set of lymphokines. Mosmann and colleagues (1986) extended these observations and proposed the existence of two subsets of helper T cell (Th cell) clones in the mouse (for review see Mosmann and Coffman, 1989). The first subset, Thl, produces IL-2 and IFN-y, whereas the second subset, Th2, fails to produce these two lymphokines. Instead, Th2 secretes a TCGF activity distinct from IL-2, a MCGF activity distinct from IL-3, an IgE enhancing activity, an IgA enhancing activity and an eosinophil-CSF activity. The first three are the functions of IL-4 whereas the last two are the functions of IL-5. These two Th cell subsets influence the outcome of an antigenic

6820 1650 S

ND ND ND

II

428 599 S

143 363 S

II

I

3.3 230 361 S

I

0 179 577 S IL-3 (rig/mE)

ND ND 0 ND

IL-2 (U/mE)

0 0 7078 S

0 31 27 S

II

ND ND ND

I

38 82 S

II

IL-4 (rig/mE)

ND ND 0 ND

I

IL-4 (ng/mL)

7.1 5.6 24.3 IS

II

ND ND ND

I

15 149 IS

II

IL-5 (ng/mL)

ND ND 0 ND

I

IL-5 (ng/mL)

8.0 4.6 16.8 IS

II

ND ND ND

I

12 15 IS

II

IL-6 (ng/mL)

ND ND 0 ND

I

IL-6 (ng/mL)

( ( i

153 4 S

I

i

GM-C~ (ng/mI

0 40 210 S

(ng/mI_ I

GM-CS

*IL-2, IL-3, IL-4, IFN-~; 6 hr stimulation; IL-5, IL-6, IL-10; 12 hr stimulation; GM-CSF, 12 hr stimulation. I: E tIL-2, IL-3, IL-4, II-6, IFN-~; 6hr stimulation; IL-5, GM-CSF, ILl0; 24hr stimulation; ND, not determin~ antiCD3 stimulation; plate coated anti-CD3 was used.

PMA/A23187 Anti CD3 CsA sensitivity

tExp2

*Expl PMA A23187 PMA/A23187 CsA sensitivity

II

I

I

II

IL-3 (ng/mL)

IL-2 (U/mL)

TABLE 1. Requirements of Stimulations for Lymphokine Production

308

N. ARM et al. Th I

Coupting

.

Th 0

D

T

O

Q

IL-2

IL-2

IL-4

IL-4

DNA binding proteins

O

O

O

[] IL-2

,

IL-4,

11_-2 11.-4

Express Thl specific binding protein(s)

L

[]

IL-2 IL-4

[..J ~

O

~

a O O

of TCR and signaling

h

O

~

11.-2

~

IL-4

The some signaling pathway

con octivote different binding proteins which interact with IL.-2 or 11--4 genes

[]

Express only Th 2 specific binding protein(s)

FIG. 3. Models for differential expression of lymphokine genes in Thl and Th2 subsets. Upper left: Thl specific signal transduction pathway whose target is Thl specific nuclear protein(s). Both Thl and Th2 specific proteins are present. Upper middle: both Thl and Th2 specific signaling pathways are working. Upper right: Th2 specificsignaling pathway whose final target is Th2 specific nuclear protein(s). Bottom left: only Thl specific binding protein(s) are expressed. Bottom middle: the same signaling pathway interacts with different binding proteins which interact with IL-2 or IL-4 genes. Bottom right: only Th2 specific binding protein(s) are expressed.

response either toward humeral or cell-mediated immunity. Many human T cell clones produce both IL-2 and IL-4 and the existence of Th 1- and Th2-1ike subsets in humans was not clear at that time (for review see Yokota et al., 1988). However, more recent studies indicate that this dichotomy operates in the human system as well (Parronchi et al., 1991). What is the biochemical mechanism which accounts for the distinct patterns of lymphokine production observed in the two subsets? We considered two possibilities which are not mutually exclusive: differential signal coupling and differential expression of transcription factors (Fig. 3). According to the first model, the signal transduction pathways are different between Thl and Th2, namely, the interaction with antigen on antigen presenting cells or the coupling of the TCR with signal transduction machinery. Supporting the model, inositol phosphate and intracellular Ca 2+ concentrations are lower in Th2 than in Thl upon T cell activation (Gajewski et al., 1990). This suggests the involvement of a signaling system other than the PKC pathway utilized in Thl. Furthermore, PGE2, forskolin and cholera toxin, known to elevate cAMP levels, inhibit IL-2 and IFN-7 production but not IL-4 and IL-5 production in helper T cells (Betz and Fox, 1991; Munoz et al., 1990). We also confirmed the effects of PGE2 using mouse Thl (DI.I) and Th2 (D10) clones. Interestingly, PGE2 inhibits the expression of IL-3 and GM-CSF in Thl clone but not in Th2 clone (Y. Naito, unpublished observation). Alternatively, according to the second model, Thl and Th2 clones differ in the final targets of the signaling pathway by expressing unique sets of trans-activators and/or DNA binding proteins which recognize eis-acting regulatory sequences of cytokine genes. It is tempting to speculate that Thl and Th2 express Thl-specific and Th2-specific transcription factors, respectively. For example, an 11 base pair cis-acting P element of the IL-4 gene (Abe et al., 1992) shows homology with the regulatory region of the IL-5 gene but not with that of the IL-2 or IFN-7 gene, The protein which interacts with the P element may be attractive as a candidate for Th2-specific factors. It is also possible that, beside the positive regulator, Thl and Th2 may differ in the expression of a negative regulator which interacts with the inhibitory sequence of the upstream of the IL-4 gene (Li-Weber et al., 1992).

Lymphokine genes in T cells

309

GGAAAGTCCCC

GM.CSF-SS

,c,c,po,,o,,cccccoccqccc-73 t

t

1

J

GM2

GC box

|(NF-JrB?) I

(~pi?) I Constitutive

Inducibte

foctor

~

PMA

m RNA

. ~

Co+* Ionophore

FIG. 4. Schematic representation of mouse GM-CSF regulatory region.

The two Th subsets are believed to be differentiated from a common precursor Thp, which is derived from naive T cells which produce only IL-2 upon stimulation by antigen on antigen presenting cells. It appears that the direction of differentiation is determined by cytokines in the microenvironment (Ben-Sasson et aL, 1990; Swain, 1991). The Thp clone differentiates into Thl when IFN- 7 is supplied in the medium, while the Th2 clone develops when the culture contains IL-4. Whether this is due to selective proliferation of a particular clone or due to the switch of the expression pattern in individual cells remains to be determined.

4. REGULATION OF THE EXPRESSION OF THE GM-CSF GENE: INVOLVEMENT OF ONCOGENES AS TRANSCRIPTION ACTIVATORS To characterize a series of biochemical events initiated by antigen stimulation, we have established the following goals using the GM-CSF gene as a model. (1) Define the regulatory regions of lymphokine genes that mediate the response to T cell activation signals or viral trans-activators. (2) Characterize the proteins which interact with the regulatory sequences of lymphokine genes. (3) Examine the functions of these proteins using transcription assays/n vitro. We have found that transfected mouse GM-CSF gene is activated by a combination of PMA and A23187 in human T ceU leukemia Jurkat cell line and we have previously identified a c/s-acting region spanning the GM-CSF promoter (positions - 9 5 to +27) that confers inducibility to reporter genes in a transient transfection assay (Miyatake et al., 1988b). Further analysis identified three elements required for efficient induction, referred to as GM2, GC-box and CLE0. GM2 defines a binding site for protein(s) whose binding is inducible by PMA and the GC-box is a binding site for constitutively bound proteins (Fig. 4). 4.1. GM2 DEFINESAN ACTIVATION-DEPENDENTBINDINGSITE FOR NF-GM2 Point mutational analysis defined two functional sites (GM2 and GC box) within CLE2 ( - 9 4 to - 8 8 ) and GC box element. Changing the G at position - 9 1 to A and the T at - 8 3 to A abolished the ability to compete with wild type oligonucleotides for the binding of NF-GM2 without affecting the binding of other constitutive factors to the GC-box. On the other hand, an oligonucleotide having mutation at - 77 retained the ability to interact with NF-GM2 but not with GC-box binding proteins (AI, A2 and B). The GM-CSF templates with these mutations are inactive in a transcription assay, indicating that the binding ability correlates well with transcription activity (Sugimoto et aL, 1990). Thus these DNA binding proteins may play a role in inducing transcription of the GM-CSF gene. The decameric GM2 sequence (GGTAGTTCCC) has a 7-base homology with the mouse Igg enhancer (GGGAATCCCC, the opposite direction of the canonical ~cB motif; for reviews see Baeuerle,1991; Blank et al., 1992). Indeed, addition of the Ig~cB sequence abolished NF-GM2 complex formation in oligonucleotide competition experiments by using the GM2 motif. Thus we tentatively concluded that the NF-GM2 protein is similar to the NF-~cB

310

N. ,Moa et ai.

protein and is probably a member of the NF-xB protein family. The NF-GM2 protein purified from activated Jurkat cells based on the binding activity to the GM2 sequence shows characteristics similar to NF-xB protein (Tsuboi et aL, 1991). Both the NF-GM2 and NFxB proteins consist of two subunits, 50 kDa and 65 kDa. NF-GM2 shares the same epitope with an antiserum against KBF1 (50 kDa subunit of NF-xB protein). The purified NFGM2 protein showed specific binding to the GM2 sequence as judged by methylation interference assay showing significant interference of the methylation of six gnanines in both strands required for NF-GM2 binding. Thus a 10 base pairs sequence from position -91 to - 8 2 of the GM2 motif of the GM-CSF promoter was defined as the binding site for the NF-GM2. The purified NF-GM2 protein, however, failed to stimulate transcription from the GM-CSF gene, yet was capable of supporting in vitro transcription from the xB enhancer, suggesting possible involvement of a protein similar to but distinct from NF-xB in GM-CSF expression. NF-xB is a transcription factor receiving much attention recently since this protein controls expression of a wide variety of genes. NF-xB activation is induced by a variety of substances, such as PMA, inflammatory cytokines (TNF-# and IL-1), agents provoking oxidative stresses (H202) (Staal et al., 1990; Droege et al., 1992) and viruses (HTLV-I, CMV, etc.) which represent a threat to cells. The activation of cytoplasmic NF-xB is thought to be mediated by its signal-dependent release from an inhibitory protein designated IxB allowing NF-xB to translocate to the nucleus and to bind to the xB element. Molecular cloning revealed that NF-xB consists of 50 kDa and 65 kDa polypeptides both belonging to the family of c-rel protooncogene products (Ghosh et al., 1990; Kieran et aL, 1990; Nolan et a1.,1991). The c-tel genes are part of a rapidly growing family of transcription factors whose recognition sequence is indistinguishable from that of the authentic NF-x B protein. In addition to the rel family proteins, there are other proteins, such as HIV-EPI/PRDII-BFI/MBP1 (Maekawa et al., 1989; Fan and Maniatis, 1990; Baldwin et al., 1990), HIV-EP2/MBP2 (Nomura et al., 1991; Rustgi et aL, 1990) and KBP-I (Rustgi et aL, 1990) which recognize the same DNA sequence motif. They are collectively called the zinc finger binding proteins. Using a southwestern technique, we have isolated several eDNA clones encoding DNA binding proteins which recognize the GM2 sequence. One is the PRDII BF-1 and the others are thought to be novel clones. One novel clone, designated pKY-Z, contains two zinc finger motifs highly homologous to the C-terminal set of Zn fingers of PRDII BF-1 but which contains quite different flanking sequences of the finger domain from those of PRDII BF-1. Northern analysis indicated that the expression of the mRNA is specific to T cells and two different mRNA species (8 and 5 kb) hybridized with the KY-Z probe (K. Yokota, unpublished results). The pKY-Z clone was found to be identical to KBP-1 whose partial amino acid sequence has been described (Rustgi et aL, 1990) and to HIV-EP3 (S. Ishii, personal communication). Interestingly, however, we failed to isolate the eDNA for NF-xB by this method, probably due to the requirement for the precursor of NF-xB (p105) to be processed before binding activity is manifested. At this moment, we cannot rule out the possibility that the NF-GM2 protein is different than the NF-xB protein. The function of each DNA binding protein, if any, in the activation of the GM-CSF promoter has to be assessed by transcription assay in vitro and/or in vivo.

4.2. THE GC-Box IS A BINDINGSITEFOR CONSTITUTIVEPROTEINS The recognition sequences of three proteins, Al, A2 and B which bind the GC-box independent of antigenic stimulation are indistinguishable, even though they are distinct proteins. Purified AI is indistinguishable from Spl as judged by the mobility shift assay (Yamaguchi-Iwai et al., 1991) and crossreacts with an antiserum against Spl. Al can be separated from A2 and B by a DEAE Sepharose column, while the latter two are coeluted in all columns tested, suggesting that A2 and B are related. Purified Al, as well as the fraction containing A2 and B, is capable of supporting transcription of the GM-CSF promoter in vitro (E. S. Masuda, unpublished observation).

Lymphokine genes in T cells 4.3. CLE0: A L n ~ B ~

311

PMA AND CALCnna SIGNALS?

Two factors, NF-CLE0a and NF-CLE0b, recognize the 3'-half and the 5'-half, respectively, of the CLE0 element (at position - 4 0 to - 5 4 ) located downstream of the CLE2/GC box (Miyatake et al., 1991). The same region was identified in the human GM-CSF gone as well (Nimer et al., 1988). The binding of NF-CLE0s to DNA is inducible and neither PMA nor calcium ionophore alone is sufficient for maximum stimulation (E. S. Masuda, unpublished observaion). NF-CLE0~ recognizes the region required for PMA/A23187 stimulation, while NF-CLE0/1 interacts with the region inhibitory for the expression of the GM-CSF gone. Depleted nuclear extracts prepared by passing through a CLE0-oligonucleotide affinity column failed to support in vitro transcription, indicating the importance of CLE0 binding protein(s) for transcription from the GM-CSF promoter (E. S. Masuda, unpublished observation). Interestingly, the CLE0 of the mouse GM-CSF gone is highly homologous to the sequences located at similar positions relative to the transcription initiation sites of the IL-4, IL-5 and G-CSF genes. The CLE0 elements of the IL-4 and IL-5 genes, but not of the G-CSF gone which is silent in T cells, are also recognized by NF-CLE0a and NF-CLE0b, suggesting that these proteins play a role in the coordinate expression of several lymphokine genes in activated T cells. The recognition sequence of NF-CLE0a contains the core motif (GGAA) of the PU box, i.e. the recognition sequence of proteins containing the Ets domain. Members of the protooncogene Ets family encode sequence-specific trans-activation factors including Ets-l, Ets-2, Erg, Elk-l, Elk-2, E74, PU1 (Karim et al., 1990) and more recently Elf-1 (Thompson et al., 1992). Tissue distribution of the known Ets proteins suggests that they regulate the expression of tissue-specific genes. For instance, PU1 is expressed in macrophage and B cells, Elk in lung and testis, Ets-1 in T cells and Elf-1 in B cells and quiescent or activated T cells. The Ets binding site, a centrally located purine-rich sequence, 5'-GGAA-3', can be found in the promoter enhancer region of a variety of T cell-specific genes including T cell tropic viruses (HTLV-1, HIV and MSV) and cell surface receptors (CD2, CD3, TCR~,/~ and ~). The Ets binding sequence motif is also found in the CLE0a region of GM-CSF ( T A G A G G A A A T G ) , IL-4 ( G G G A A A A T G A ) and IL-5 ( G A G G A A A T G A ) and in NFAT-1 ( A G G A G G A A A A A ) and NFIL-2B ( A A G A G G A A A A A ) of the IL-2 enhancer (for review see Ullman et al., 1990). Induction of NF-AT, a major target for CsA, may require two activation-dependent events: the CsA-insensitive de nero synthesis of a nuclear component and CsA-sensitive translocation of a pre-existing component involving a Ca 2+-dependent reaction (Flanagan et al., 1991; McKeon, 1991). Newly synthesized nuclear component of NF-AT appears to be the transcription factor AP-I (Jain et al., 1992) and Elf-1 interacts with NFAT as well as with NFIL-2B sequences. Thus, the E/f-l protein may regulate the IL-2 enhancer in concert with inducible factors (possibly AP-1) bound to adjacent sites of DNA. Interestingly, Ets-I and Ets-2 are phosphorylated in T cells upon antigen stimulation and calcium ionophore stimulation mimics this reaction (Fujisawa et al., 1990). Thus, it is tempting to speculate that Elf-l, which contains a serine/threonine-rich region immediately C-terminal to the basic domain, also undergoes phosphorylation in response to signals. The involvement of the Ets protooncogene family in transcription of the GM-CSF gone, however, remains to be clarified.

5. ROLE OF VIRAL T R A N S - A C T I V A T O R S

IN LYMPHOKINE GENE EXPRESSION

Viral trans-activators such as HTLV-I encoded Tax, which activates HTLV-I LTR via a 21 bp enhancer and Bovine Papilloma Virus 1 (BPV-1) encoded E2, which activates BPV genes through the Long Control Region (LCR) of the BPV genome, also activate several cytokine genes (Heike et al., 1989; Miyatake et al., 1988a). The Tax protein acts through induction of NF-lcB binding to a KB-liko sequence without TCR signaling. T cells from Adult T cell Leukemia (ATL) patients are known to produce several cytokines including GM-CSF, IL-5, IL-6 and IFN-y and to express a high level of the IL-2 receptor (IL-2R~) as well. One sequence (CLE1), which was proposed as a consensus cis-acting element for lymphokine gone induction (Miyatake et al., 1985; Stanley et al., 1985) and is located at position - 113 to - 9 6 of the mouse GM-CSF gene, responds to Tax stimulation (Miyatake et al., 1988b). A recent report indicated that the T cell surface molecule CD28 is likely to activate the IL-2 gone via the the CLEI element, since stimulation of CD28

312

N..A.at/d et al. DNA binding, dimerization

Activation domain I00 I

[i Spht

domain

200 I

, Ncot

~ , I Kpnt/Asp 718

410o.o I I BIcI. I

283 pCDSR~cE2 (Kpnt) [ 4tO

I pmGMCAT-96 StimuLation

-

pBPV-LCR- CAT +

-

+

Conversion (%) pCDSR c(

0.4

2.0

0.8

0.8

pCDSRcc-c E2 (Kpnt)

7.0

25.8

2.8

1.9

pCDSR•-cE2

I1.0

64.6

63.3

62.7

FIG. 5. E2 protein lacking DNA binding domain retains the ability to stimulate transcription from the GM-CSF promoter but not from BPV-LCR. Plasmid pCDSR~,-cE2 (KpnI) was constructed by cleaving plasmid pCDSR=-cE2 (Heike et al., 1989) with KpnI followed by treatment with T4 DNA polymerase and ligation. The plasmid pCDSR=-cE2 (KpnI) encodes a truncated E2 protein (283 amino acids) with 22 extra amino acids (shaded box). The transfection into Jurkat cells was conducted as described previously (Heike et al., 1989). The cells were stimulated with 30 ng/mL PMA and l /zM A23187 for 8 hr before harvest. Trans-activator activity was evaluated by chlorampbenicol acetyltransferase (CAT) activity, expressed as % conversion.

induces the formation of a protein complex bound to the CLE1 element (Fraser et al., 1991). Another sequence that responds to viral trans-activators has been localized to the G M 2 / G C box element ( - 9 5 to -73) of the mouse GM-CSF gene, also known as a target for PMA/A23187 stimulation. NF-GM2 forms a complex with DNA in Tax-expressing Jurkat cells without PMA stimulation (unpublished observation). Taken together, the viral trans-activators bypass TCR signaling by modifying the component of the TCR-mediated signaling pathway. This led us to speculate that Tax abolishes the requirement for TCR signaling by converting an inactive NF-K B to an active form. This notion is supported by the finding that the Tax protein interacts directly with pl05, the precursor of the p50 subunit of the NF-KB protein (Hirai et al., 1992). Activation of the GM-CSF promoter by PMA/A23187 stimulation is CLE0-dependent, whereas Tax activates the same promoter without involving the CLE0 element (N. Koyano-Nakagawa, J. Nishida, N. Arai, K. Arai and T. Yokota, submitted for publication). These results suggest that Tax operates as a bridge between the upstream enhancer binding protein (NF-GM2) and general transcription factors. E2 binds to a specific DNA sequence (ACCN6GGT) and activates transcription from BPV-LCR. Neither the CLE2/GC box nor the CLE1 motif in the GM-CSF promoter has any homology with the consensus motif of E2 binding, yet E2 activates the GM-CSF promoter in a manner similar to Tax. Furthermore, PMA/A23187 stimulation enhances the effect of E2-dependent activation of the GM-CSF promoter in a synergistic manner, whereas no stimulation is observed in the activation of BPV-LCR (Fig. 5). To address the question of whether E2 activates the GM-CSF promoter by binding directly to the GM2/GC box sequence, we constructed truncated E2 having the N-terminal 283 amino acids. Like other trans-activators such as GCN4 and Gal4, the E2 protein contains two distinct domains. The C-terminal domain is involved exclusively in DNA binding, whereas the N-terminal domain is required for trans-activation functions. The inner domain (hinge

I

2

0.5

.5

11.7

4

20.7

5

0.4

6

0.9

7

37.0

8

44.2

9

1.6

I0

47.2

II

8.5.9

12

91.6

13

84.!

FIG. 6. Tax-E2 binding domain fusion protein activates transcription from both BPV-LCR and GM-CSF promoters. Th plasmids (pCDSR~-pX--cE2-1, pCDSR~-pX-cE2-2) is as follows. The termination codon of plasmid pCDSR~-pX (TGA t a KpnI or Asp718 site (TGG TAC CGT) by in vitro mutagenesis (pCDSR~-pX-mut). Plasmid pCDSR~-pX--cE2-1 HindlII-Asp718 fragment of plasmid pCDSRc,--cE2 with the HindlII-Asp718 fragment of plasmid pCDSR~-pX-m pCDSR~-pX-cE2-2, plasmid pCDSRcc-pX-mut was cleaved with KpnI followed by filling in with DNA polymerase I I~ fragment of plasmid pCDSR~-cE2 was replaced with the HindlII-KpnI fragment of plasmid pCDSR~-pX-mut after treal polymerase and Klenow fragment, respectively. Jurkat cells were transfected as described previously (Heike et al., 1989) 1-8; and 1, 2; pCDSR~-pX--cE2-1, 3, 4; PCDSR~-pX-cE2-2, 5, 6; pCDSR~-pX, 7, 8; pCDSR~-cE2. 9-16; transfected pCDSR~pX--cE2-1, 11, 12; pCDSR~-pX-cE2-2, 13,14; pCDSR~-pX, 15, 16; pCDSR~-cE2. 2, 4, 6, 8, 10, 12, 14 and 16 are for 8 hr before harvest.

? :

i

0.4

Conversion (%)

314

N. ARAI et al.

region) may confer flexibility on the E2 protein (Giri and Yaniv, 1988). E2 lacking its DNA binding domain (pCDSR~t-cE2(KpnI)) retained the ability to activate pmGM-CAT96 with or without PMA/A23187 stimulation, albeit at a lower level (Fig. 5). In contrast, activation of the BPV LCR was virtually abolished by removing the DNA binding domain of E2. A chimeric protein (pCDSR~-pX-cE2-2) that contains Tax and the DNA-binding domain of E2 at N- and C-terminus, respectively, stimulates transcription from BPV-LCR in a manner similar to E2, whereas pCDSR~t-pX-CE2-1 having Tax and the DNA binding site of E2 but lacking the hinge region is inactive (Fig. 6) (T. Heike, unpublished). This indicates that the function of the activation domain of E2 can be replaced by Tax despite no obvious sequence homology between the two proteins. Therefore, E2 activates various promoters in at least two ways. A direct mechanism recognizes the consensus DNA motif via its DNA binding domain resulting in the stimulation of the E2 activator domain. In addition, an indirect mechanism interacts with separate sequencespecific DNA binding protein(s). NF-~cB targets Tax to the promoter via the r B element in a way similar to Oct-l, which targets HSV-1 encoded VP-16 to its respective promoter. Judging from the similarity between the actions of E2 and Tax, the NF-rB-like protein (NF-GM2) might also target E2 via the GM2 sequence to activate the GM-CSF promoter. It should be noted, however, that the G C box element contained within the mouse GM-CSF promoter is recognized by three proteins, including Spl. In view of the physical interaction between Spl and E2 (Li et al., 1991), it is also possible that E2 activates the mouse GM-CSF promoter by interacting with Spl. In summary, several cellular factors are likely to sequester Tax or E2 to the specific DNA site through protein-protein interaction to exert their trans-activator functions; however, the detailed mechanism remains to be investigated. A.

Modet for TCR mediated signal tronsduction TCR

ModeL I Signal 2 (Ca2+)

Signal I (PKC)

Tax

E2 "'~ ~l

i En.ance,, I

I Enhance'']

Signal I

Signal 2

Model II

(PKC)

(Co 2+)

.x E2 ~

J Enhoncerl J

Model III

,°xl Signor )

Signal 2

(PKC)

E2 -~

J Enhancer 2J

(Co 2+)

0 New synthesis

FIG. 7(a). Caption opposite.

1 1

Lymphokine genes in T cells B.

315

Model for arrangementof ¢is elements in tymphokinegenes

Model I

,~,Enhoncer I I

I Enhancer~

Model 2

- ~ EnhancerI [

IEnhan~

ModeL 3

.'~JEnhan~

[Enhancer2 I

Model 4

~L

Cel.ksRencer type specificj~ I EnhancerI I

[ Enhancer2 t

FIG. 7. Roles of multiple c/s-acting elements that respond to T cell activation signals. (A) Model for TCR mediated signal transduction. (B) Model for arrangement of c/s elements in lymphokine genes. The shaded circle represented a hypothetical c/s-acting element which is recognized by cell type or developmental stage-specificactivator protein(s). The dotted circle indicates a hypothetical c/s-acting element whose binding protein is removed in certain cell types or development stage-specificprotein(s) to activate the lymphokine gene.

6. CONCLUSION AND SPECULATIONS An important aspect of the regulation of lymphokines is the gene organization on the chromosome. Several lymphokine genes such as IL-3, GM-CSF, IL-4 and IL-5 are located as a cluster on human chromosome 5 at bands 5q23-32, known to be deleted [del(5q)] frequently in patients with myeloid disorders (Le Beau et al., 1989). Physical linkage studies of the IL-4 and IL-5 genes by pulse field gel electrophoresis estimated the maximum distance that separates these genes as 310 kb. In addition, the genes encoding GM-CSF and IL-3 have been shown to be closely linked (only 9 kb apart from each other). Many lymphokine genes are composed of 4 or 5 exons. Within each lymphokine gene, the nucleotide sequence of the Y-upstream region is most highly (75-90%) conserved between species, suggesting that this region plays an important regulatory role. We have searched for common features of several lymphokine genes coordinately expressed upon antigenic stimulation and have found several short sequence motifs including CLE0, CLE1 and CLE2. An important feature of T cell lymphokine genes is their two-signal (pKc and calcium ion) dependence for expression. Functional analyses employing transfection assays have established that these cis-acting elements of lympholdne genes are required either alone or in combination for inducible expression. What is the role of each c/s-acting element in responding to activation signals and in determining cell-type specific expression? Studies of eukaryotic gene expression thus far indicate that only a limited number of regulatory proteins control a myriad of genes in mammalian cells. It appears that selective (stage-, cell type-, or induction-specific) expression is achieved by combinatorial arrangement of c/s-acting elements and the type of interactions between the proteins that bind to these elements. It is tempting to speculate that lymphokine promoters are composed of at least three (or more) distinct cis-acting elements which regulate the response to PMA or calcium signals and which confer cell-type specificity (Fig. 7). At least three elements, CLE2/GC and CLE0 which interact with inducible and constitutive proteins, are involved in the optimal induction of the GM-CSF gene. Likewise, IL-2 expression depends on multiple elements including NF-AT, NF-KB, AP-1, NFIL-2A (Oct-1 with OAP40, Ullman et al., 1991) and NF-AT (the target of CsA inhibition) which may confer inducibility and T cell specificity. The fact that the expression of the IL-2 or GM-CSM gene depends on de nova protein synthesis since anisomycin (or

316

N. ARAI et al.

cycloheximide) blocks synthesis of both mRNAs adds additional complexity. These results suggest that some early response genes play a role in the regulation of lymphokine genes. N F - A T is comprised o f a newly synthesized nuclear subunit (possibly AP-1; Jain et al., 1992) and a pre-existing cytoplasmic subunit translocated to the nucleus (Flanagan et al., 1991). The regulation of the G M - C S F gene depends on the PMA-induced binding of N F - G M 2 to the G M 2 / G C element. In addition, newly synthesized protein has to interact with the CLE0 element along with the subunit which may be a final target of the Ca 2+ signal. Thus, combination of actions of multiple c/s-acting elements yield a variety of expression patterns of lymphokine genes. It should be noted that tandem constructs of a single cis-acting element such as (•B)n, (GM2)n or (CLE0)n are not equivalent to the constructs composed of single copies of more than one type of cis-acting element such as r B - C L E 0 or GM2/GC-CLE0. The latter requires both PMA and calcium signals whereas tandem constructs may require only a partial signal. However, the molecular mechanism by which single or multiple combinations of c/s-acting elements enhance transcription in response to one or more signals remains to be clarified.

REFERENCES ABE, E., DE W ~ MAL~YT, R., MATSUDA,I., Asia, K. and AR~, N. (1992) An 11-base-pair DNA sequence motif apparently unique to the human interleukin 4 gene confers responsiveness to T-cell activation signals. Proc. hath. Acad. Sci. U.S.A. 89: 2864-2868. ARM, K., LEE, F., MIYAJIMA,A., MIYATAKE,S., ARAI, N. and YOKOTA,T. (1990) Cytokine: coordinator of immune and inflammatory responses. A. Rev. Bioehem. 59: 783-836. BALDWIN,A. S., LECL~aR,K. P., SINGH,H. and SHARP,P. A. (1990) A large protein containing zinc finger domains binds to related sequence elements in the enhancers of the class I major histocompatibility complex and kappa immunoglobulin genes. Molec. Cell Biol. 10: 1406-1414. BArnaCLE,P. A. (1991) The inducible transcription activator NF-rB: regulation by distinct protein subunits. Biochim. biophys. Acta. 1072: 63-80. BEH-SASsoN,S. Z., GRos, G. L., CONRAD,D. H., FINKELMAN,F. D. and PAUL,W. E. (1990) Cross-linking Fc receptors stimulate splenic non-B, non-T cells to secrete interleukin 4 and other lymphokines. Proc. hath. Acad. Sci. U.S.A. 87: 1421-1425. Blrrz, M. and Fox, B. S. (1991) Prostagiandin E2 inhibits production of Thl lymphokines but not of Th2 lymphokines. J. Immun. 146: 108-113. Bt~a~K, V., KOURI~KY, P. and I s v , ~ , A. (1992) NF-~cB and related proteins: rel/dorsal homologies meet ankyrin repeats. TIBS 17: 135-140. DOWNWARD,J., GRAWS,J. D., WARt,~, P. H., RAYTER,S. and CAt~TP,J~LL,D. A. (1990) Stimulation of p21 ras upon T-cell activation. Nature 346: 719-723. DROEGE,W., Eft:, H.-P. and MtI-IM, S. (1992) HIV-induced cysteine deficiency and T-cell dysfunction~a rationale for treatment with N-acetylcysteine. Immun. Today 13: 211-214. FAN, C.-M. and MANIATIS,T. (1990) A DNA-binding protein containing two widely separated zinc finger motifs that recognize the same DNA sequence. Genes Dev. 4: 29-214. FLANAGAN,W. M., COg~SY, B., BRAM,R. and CP,A B ~ , G. R. (1991) Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 352: 803-807. F ~ , s ~ , J. D., IRVING,B. A., CRABTlU~,G. R. and WEiss, A. (1991) Regulation of interleukin-2 gene enhancer activity by the T cell accessory molecule CD28. Science 251: 313-316. FUJISAWA,S., KOIZUMI,S., FISHER,R. J., BHAT,N. K. and PAPAS,T. S. (1990) Phosphrylation of the ETS-2 protein: regulation by the T-cell antigen receptor-CD3 31 complex. Molec. Cell Biol. 10: 1249-1253. GgrewsKi, T. F., SCHELL,S. R. and FITCH,F. W. (1990) Evidence implicating utilization of different T cell receptor-associated signafing pathways by Thl and Th2 clones. J. Immun. 144: 4110-4120. GHOSH,S., Gn'FO~, A. M., ~ , L. R., T~UPST,P., NOLAN,G. P. and BALTIMORE,D. (1990) Cloning of the p50 DNA binding subunit of NF-rB: homology to rel and dorsal. Cell 62: 1019-1029. Gala, I. and YAmv, M. (1988) Structural and mutational analysis of E2 trans-activating proteins of papillomaviruses reveals three distinct functional domains. EMBO J. 7: 2823-2829. I-I~KE, T., M~YATAr~,S., YOSHn3A,M., ARAI, K. and ARAI, N. (1989) Bovine papilloma virus encoded E2 protein activates lymphokine genes through DNA elements, distinct from the consensus motif, in the long control region of its own genome. EMBO J. 8: 1411-1417. Hat,J, H., FUJISAWA,J., SUZUKI,T., UreA, K., MURAMATSU,M., TSUBOI,A., AR~, N. and YOSHIDA,M. (1992) Transcriptional activator tax of HTLV-1 binds to the NF-rB precursor p105. Oncogene 7: 1737-1742. Im3ODEN,J. B., SHOBACI¢,D. M., PATTISON,G. and STOeO,J. D. (1986) Cholera toxin inhibits the T cell antigen receptor-mediated increases in inositol trisphosphate and cytoplasmic free calcium. Proc. hath. Acad. Sci. U.S.A. 83: 5673-5677.

Lymphokine genes in T cells

317

JAIN, J., M c C o Y , G., VALGE-ARcmm,V. E. and RAO,A. (1992) Nuclear factor of activated T cells contains Fos and Jun. Nature 356: 801-804. KAmM, F. D., URNESS,L. D., TmmMM~L,C. S., KLEMSZ,M. J., McK~L'HFm, S. R., CELAD^,A., VAN BEVEI~.m C., MAXI,R. A., GUNTHER,C. V., NYE,J. A. and GRAVES,B. J. (1990) The ETS-domain: a new DNA-binding motif that recognizes a purin-rich core DNA sequence. Genes Dev. 4: 1451-1453. KnmAN, M., BLANK,V., LO~EAT,F., V^N-DEmmCI~OW, J., LoTrsPEICH,F., LE BAIL,O., URnAN, M. B. and ISlU~L, A. (1990) The DNA binding subunit of NF-~B is identical to factor KBFI and homologous to the tel oncogene product. Cell 62: 1007-1018. KLAUS)~-ER,R. D. and SAM~tSON,L. E. (1991) T cell antigen receptor activation pathways: the tyrosine kinase connection. Cell 64: 875-878. LE BEAU,M. M., LEMONS,R. S., ESPINOSA,R., LARSON,R. A., ARAI,N. and ROWLEY,J. D. (1989) Interleukin-4 and interleukin-5 map to human chromosome 5 in a region encoding growth factors and receptors and are deleted in myeloid leukemias with a del(Sq). Blood 73: 647-650. LI, R., KNIGHT,J. D., JACKSON,S. P., THAN,R. and BOTCHAN,M. R. (1991) Direct interaction between the BPV enhancer E2 protein mediates synergistic activation of transcription. Cell 6~: 493--505. LI-WEnm, M., EDER,A., ~ - C z E P A , H. and KRAMMim,P. H. (1992) T cell-specific negative regulation of transcription of the human cytokine IL-4. J. Immun. 148: 1913-1918. MAEKAWA, T., SAKURA,H., SUDO, T. and ISHII, S. (1989) Putative metal finger structure of the human immunodeficiency virus type 1 enhancer binding protein HIV-EP1. J. biol. Chem. 264: 14591-14593. McKEoN, F. (1991) When worlds collide: immunosuppressants meet protein phosphatases. Cell 66: 823-826. MIY^TAKE,S., OTSUKA,T., YOKOTA,T., LEE,F. and ARM, K. (1985) Structure of the chromosomal gene for granulocyte-macrophage colony stimulating factor: comparison of the mouse and human genes. EMBO J. 4: 2561-2568. MIYATAKE,S., SEIKI,M., DE WAALMALErYT,R., HEIr,E, T., FU~ISAWA,J., TAKEBE,Y., NlSmDA,J., SHLOMAI,J., YOKOTA,T., YosmDA, M., Al~L K. and ARAI,N. (1988a) Activation of T cell-derived lymphokine genes in T cells and fibroblasts: effects of human T cell leukemia virus type I p40x protein and bovine papilloma virus encoded E2 protein. Nucleic Acids Res. 16: 6547-6566. MIYATAKE,S., SEIKI, M., YOSmDA, M. and AR~a, K. (1988b) T-cell activation signals and human T-cell leukemias virus type I-encoded iM0tax protein activate the mouse granulocyte-macrophage colony-stimulating factor gene through a common DNA element. Molec. Cell Biol. 8: 5581-5587. MIY^TAKE,S., SHLOMAI,J., ARAI, K. and ARALN. (1991) Characterization of the mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter: nuclear factors that interact with an element shared by three lymphokine genes-those for GM-CSF, Interleukin-4 (IL-4) and IL-5. Molec. Cell Biol. 11: 5894-5901. MOSMANN,T. R. and CO~MAN, R. L. (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. A. Rev. lmmun. 7: 145-173. MOSMA~N,T. R., CHEItWXNSKI,H., BOND,M. W., GmDHN, M. A. and COFFMANN,R. L. (1986) Two types of murine helper T cell clone: I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immun. 136: 2348-2357. MUNOZ, E., ZImlAGA,A. M., MEllOW, M., SAUTEIt,N. P. and HUBER,B. T. (1990) Cholera toxin discriminates between T helper 1 and 2 cells in cell receptor-mediated activation: role of cAMP in T cell proliferation. J. exp. Med. 172: 95-103. MtmAMATSU, M., KAmucm, K. and Axe, K. (1989) A protein kinase C cDNA without the regulatory domain is active after transfection/n vivo in the absence of phorbol ester. Molec. Cell Biol. 9: 831-836. NIM~R, S. D., MOmT^ E. A., MAI~TIS,M. J., WACHSMAN,W. and GASSON,J. C. (1988) Characterization of the human granulocyte-macrophage colony-stimulating factor promoter region by genetic analysis: correlation with DNase I footprinting. Molec. Cell Biol. 8: 1979--1984. NOLAN,G. P., GHOSH,S., LIou, H-C., TEMPT, P. and BALTIMORE,D. (1991) DNA binding and IKB inhibition of the cloned p65 subunit of NF-~B, a rel-related polypeptide. Cell 64: 961-969. NOMURA,N., ZHAO,M-J., NAGASE,T., MAEKAWA,T., IsmzAIO, R., TABATA,S. and ISHII,S. (1991) HIV-EP2, a new member of the gene family encoding the human immunodeficiency virus type 1 enhancer-binding protein; comparison with HIV-EP-1/PRDII-BF1/MBP-1. J. biol. Chem. 266: 8590-8594. PARRONCm, P., MACCHIA, D., PICCINNI, A-P., BlSWAS, P., SIMONELLI)C., MAGGI, E., RICCI, M., ANSARI, A. A. and ROMAGNANLS. (1991) Allergen- and bacterial antigen-specific T-cell clones established from atopic donors show a different profile of cytokine production. Proc. hath. Acad. Sci. U.S.A. 88: 4538-4542. RAZANA~AONA,D., M AROC,C., LOPEZ,M., MANNOIm,P. and GAnERT,J. (1992) Shift from post-transcriptional to predominant transcriptional control of the expression of the GM-CSF gene during activation of human jurkat cells. Cell Growth Diff. 3: 299-305. ROST(3L A. K., VAN'T VEER, L. and BERNARDS,P,. (1990) Two genes encode factors with NF-)cB- and H2TFI-like DNA-binding properties. Proc. natn. Acad. Sci. U.S.A. 87: 8707-8710. SHAw, G. and KAMEN,R. (1986) A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46: 659-667. STAAL,F. J., P,OEDEmm, M., I-I~z~BEaG, L. A. and HERZENnERG,L. A. (1990) Intracellular thiols regulate activation of nuclear factor ~ B amd transcription of human immunodeficiency virus. Proc. natn. Acad. ScL U.S.A. 87: 9943-9947.

318

N. ARAI et al.

STANLEY,E., METCALF,D., SOBI~ZCZUK,P., COUGH,N. M. and ~ , A. (1985) The structure and expression of the murine gene encoding granulocyte-macrophage colony stimulating factor: evidence for utilization of alternative promoters. EMBO J. 4: 2569-2573. SuGn~oro, K., Tstmol, A., MIY^T^KE,S., Ag~, K. and ARAI, N. (1990) Inducible and non-inducible factors co-operatively activate the GM-CSF promoter by interacting with two adjacent DNA motifs. Int. natn. lmmun. 2: 175-182. SWAIN,S. (1991) Regulation of the development of distinct subsets ofCD4 + T cells. Res. lmmun. 142: 14-18. THOMPSON,C. B., WANO,C.-Y., Ho, I.-C., BOHJANeN,P. R., PETRY~UAX~,B., JUNE,C. H., MIESFELDT,S., ZHANG, L., NABeL, G., KAltPISSrd, B. Y. and L~DBN, J. M. (1992) c/s-Acting sequences required for inducible interlenkin-2 enhancer function bind a novel Ets-related protein, Elf-l. Molec. Cell Biol. 12: 1043-1053. TSUBOI,A., SUGIMOTO,K., YODOI,J., MIYATAKE,S., ARAI, K. and ARt.I, N. (1991) A nuclear factor NF-GM2 that interacts with a regulatory region of the GM-CSF gene essential for its induction in response to T-cell activation: purification from human T-cell leukemia line Jurkat cells and similarity to NF-~B. Int. natn. Immun. 3: 807-817. ULLMAN,K. S., NORTHROP,J. P., VERWFJJ,C. L. and CRAnTR~, G. R. (1990) Transmission of signals from the T lymphocyte antigen receptor to the genes responsible for cell proliferation and immune function: the missing link. A. Rev. lrnmun. 8: 421--452. ULLMAN,K. S., FL^N^GAN, W. M., EDWARDS,C. A. and C-'RAnTIU~,G. R. (1991) Activation of early gene expression in T lymphocytes by Oct-1 and an inducible protein, OAP40. Science 254: 558-562. Y ~ o u c m - I w ~ , Y., TSUBOI,A., MIY^TAKE,S. and ARAI, N. (1991) Constitutive DNA binding factor(s) essential for induction of the GM-CSF gene by antigen stimulation in Jurkat cells. J. Cell Biochem. 15B (Suppl.): 206. YOKOTA, T., ARAI, N., DE VRW~S,J., SPITS, H., BANCHEREAU,J., ZLOTNIK,A., RENNICK, D., HOWARD, M., TAKEaE,Y., MIYATAKE,S., L~, F. and AsTd, K. (1988) Molecular biology of interlenkin 4 and interleukin 5 genes and biology of their products that stimulate B cells, T cells and hemopoietic cells. Immun. Rev. 102: 138-187.