transcriptional regulation of the il-2 gene

Transcriptional regulation of the IL-2 gene Jugnu Jain, Christine Loh and Anjana Rao

Dana-Farber Cancer Institute and Harvard Med ica l School, Boston, USA

The past two years have seen significant advances in our understanding of IL-2 gene transcription. Many of the relevant transcription factors have been identified, the intracellular mechanisms regulating their functions are being elucidated, and the multiple roles of calcineurin are beginning to be

appreciated.

Current Opinion in Immunology 1995, 7:333-342

Introduction

The cytokine IL-2, a powerful growth factor for a variety of immune system cells, is produced by T cells activated with antigen [1]. IL-2 mP.NA is not present in resting CD4 + T cells, but can be detected within 30 minutes of their stimulation [2]. In this review, we will focus on the structure and regulation of the nuclear factors implicated in IL-2 gene transcription, with emphasis on the role of calcineurin and the mechanisms of immunosuppression by cyclosporin A (CsA) and FK506. CD28-mediated costimulation of IL-2 production will also be discussed briefly.

Control of IL-2 mRNA levels

The steady-state levels of IL-2 mR.NA achieved in stimulated T cells represent a balance between transcription and degradation, with the kinetics of induction being determined by the rate of transcriptional activation and the kinetics of decline by the rate of degradation (Fig. 1). The results of nuclear run-on assays, DNase I hypersensitivity studies, and in vivo footprinting are all consistent with a transcriptional mechanism for IL-2 mP.NA induction [3-6,7°°]. IL-2 gene transcription appears to depend on the continued presence of a stimulus [8]. In general, maximal steady-state levels of IL-2 mP.NA are achieved at four to eight hours following exposure of the T cells to stimulus; the dechne is more variable, but background levels are usually approached by about 24 hours [2,8,9°°]. The decline in IL-2 mR.NA levels occurs considerably faster than the dechne in the rate of IL-2 gene transcription [8,9°°], and may reflect the induction of a mechanism dependent on protein synthesis for the selective degradation of cytokine mP.NAs [4,8,9°°,10,11].

The question of how long transcription proceeds in the continuing presence of a stimulus has not been adequately addressed. Depending on the cell line and stimulus used, transcription peaks at 12-24 hours

following stimulation, and dechnes only slowly thereafter [4]. In vivo footprinting indicates that the regulatory elements on the IL-2 promoter remain occupied for as long as 11 hours following exposure to stimulus [7°°]. Thus it is possible that under physiological conditions, transcription continues (perhaps at a reduced level) for as long as the antigen is present. Decline of transcription may reflect decreases in the levels of activating transcription factors, their dissociation from binding sites on the promoter secondary to post-translational modification, or their replacement by related proteins that lack (or repress) transcriptional activity (see Fig. 1).

An inducible degradative mechanism [9°°,10,11] may control the levels of cytokine mR.NA in activated cells. Treatment of activated T cells with cycloheximide and other protein synthesis inhibitors results in a remarkable accumulation of IL-2 m_R.NA without affecting ongoing IL-2 transcription [4,8], suggesting the involvement of a degradative process that is dependent on protein synthesis. Treatment with actinomycin D considerably stabilizes cytokine mR.NAs [8,9°°,12], suggesting that the degradative mechanism is induced at the level of P.NA synthesis. The costimulatory CD28-B7 pathway augments IL-2 production at least partly by inhibiting the selective degradation of cytokine mP.NAs ([9°°,10]; see below).

Regulatory sequence elements of the IL.2 gene

When tested by transient transfection in vitro, IL-2 promoter constructs containing ~300bp 5' of the transcription start site were sufficient to mediate T-cell specific, inducible transcription of a linked reporter gene (reviewed in [13°]; see Fig. 2). The human and murine IL-2 promoters show extensive sequence similarity for an additional 300 bp beyond this region [14], and these distal sequences enhance promoter activity [14-16]. Strong positional effects in transgenic mice, however, suggest that longer range interactions control IL-2 gene

Abbreviations AP-l--activating protein-l; bp~base pair; CsA--cyclosporin A; IL--interleukin; JNK--Jun amino-terminal kinase; kb~kilobase;

NFAT--nuclear factor of activated T cells.

© Current Biology Ltd ISSN 0952-7915 333

334 Lymphocyte activation and effector functions

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Fig. I . Kinetics of induction of the IL-2 gene and of the major transcription factors thought to regulate its transcription. The time scale is approximate (and different in each panel) as data from several studies, using different cell lines and conditions of stimulation, have been combined. Dashed lines and arrows are used to indicate that experimental data for these time points are not readily available. The top left panel shows that the steady-state level of IL-2 mRNA declines more rapidly than the rate of IL-2 gene transcription. This appears to be due to the induction of a selective mechanism (dependent on RNA and protein synthesis) for degradation of cytokine mRNAs (arrow). The other panels depict the kinetics of the induction of mRNAs encoding relevant transcription factors, and the kinetics of appearance of other transcription factors in the nucleus. NFATc and Oct-2 mRNAs are expressed at low levels in resting cells [24",49].

transcription in vivo [17°]. Only one of 17 transgenic lines containing 583 bp of the IL-2 promoter expressed a linked reporter gene in an inducible, tissue-specific fashion, indicating that this region of the promoter lacks the 'locus control' sequences required for position-independent, copy number dependent expression [18].

The bulk of the information on regulatory elements derives from studies of the 300bp region upstream of the transcription start site (termed here the IL-2 promoter). In this review, we focus on the known transcription factors nuclear factor of activated T cells (NFAT), Oct, activating protein-1 (AP-1) and NFKB, which bind to identified positive elements in the IL-2 promoter (Fig. 2).

also Note added in proo0. All were originally cloned from T cell cDNA libraries, and thus may play a role in IL-2 gene transcription. NFAT3 mR.NA, however, is expressed predominantly outside the immune system and NFATx/NFAT4 mRNA is expressed at higher levels in the thymus than in the periphery ([25°°]; T Hoey, personal communication). NFATp is expressed at high levels in resting T cells [26], whereas NFATc is induced following activation [24°'], suggesting that NFATp regulates an early stage, and NFATc a later stage, of IL-2 gene transcription (Fig. 1). Although the specific family member has not been identified, a defect in NFAT structure or regulation may underlie the multiple defects in cytokine production observed in a patient with severe combined immunodeficiency [27].

NFAT The lL-2 promoter contains two binding sites for members of the NFAT family of transcription factors (reviewed in [13",19,20"]; see Fig. 2). Mutation of both sites is required to eliminate IL-2 promoter function [21,22], and in vivo footprinting indicates that both sites are occupied in stimulated T cells [7"°].

At least four NFAT-family proteins (NFATp, NFATc, NFAT3, and NFATx/NFAT4) and several splice variants have been described ([23"-25°']; T Hoey, S Ya-Lin, K Williamson, X Xu, ,personal communication; see

The DNA-binding domains of the NFAT proteins are highly homologous, and distantly resemble the DNA-binding domains of members of the Rel family of transcription factors ([19,25",28°']; T Hoey, personal communication). Notably, the sequence that aligns with the sequence of the DNA-recognition loop of p50 NFKB [29°,30 °] is used for DNA recognition by NFAT-family proteins as well [28°']. Unlike Rel-family proteins, which bind as obligate dimers and make only major groove contacts, NFATp and NFATc bind as monomers to the IL-2 promoter NFAT site ([31]; T Hoey, personal communication) and contact DNA

Transcriptional regulation of the IL-2 gene lain, Loh and Rao 335

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© 1995 Current opinion in Immunology

Fig. 2. A schematic representation of the murine IL-2 promoter indicating the regulatory elements governing gene transcription. The sequence and spacing of the elements is well conserved between the human and murine IL-2 promoters [7"°]. The numbers indicate distance (in base pairs) from the major transcription start site (arrow). Filled circles denote guanine residues that are protected by in vivo footprinting in stimulated EL4 T cells, open circles indicate protected guanine residues on the opposite (non-coding) strand, and asterisks denote hypersensitive bases [7"']. The 'AP-I' site adjacent to the NFAT site is a non-consensus AP-1 site to which Fos and Jun are recruited by NFAT-family proteins. The elements denoted by boxes at -300, -226, -176 and -63 bp are also protected in vivo [7"]. A negative regulatory element that binds a zinc finger protein [110] is not shown as it is poorly conserved between the murine and human promoters and is not detected by in vivo footprinting.

partly in the minor groove (L Chen, GL Verdine, personal communication).

A characteristic feature of NFAT proteins is that they bind cooperatively with Fos- and Jun-family proteins to the distal IL-2 promoter NFAT site ([25"',32°',33]; T Hoey, personal communication; reviewed in [20"]). An extensive alteration of the nonconsensus AP-1 site within the distal NFAT site (see Fig. 1) eliminates transcription driven by the IL-2 promoter even though the proximal NFAT site is intact [22]. Overexpression of most Fos and Jun family members augments NFAT- driven transcription [34,35,36°'], whereas expression of a dominant-negative Jun inhibits this process [37"]. Although there is little evidence as yet for selective cooperation of individual NFAT-family proteins with specific Fos-Jun dimers [35,36"',38-40], it is plausible that different combinations of NFAT and Fos-Jun dimers mediate distinct functions at different cytokine sites.

Several nuclear factors have been described that are unrelated to NFAT-family proteins but that are capable of binding to the NFAT site. These include Elf-1 (an Ets-family protein [21]), interleukin binding factor (ILF) (a protein isolated by Kgt11 expression cloning through its ability to bind an NFAT oligonucleotide [41]), and NF45 and NF90 (two polypeptides purified by affinity chromatography using a mutated NFAT oligonucleotide [42]). In general, these proteins do not exhibit the properties expected ofNFAT: CsA- and FK506-sensitive appearance in nuclear extracts from appropriately stimulated T cells; sequence-specific binding to the NFAT site; and cooperative binding with Fos and Jun. They could, however, modulate IL-2 gene transcription, a possibility that was recently suggested for Ets-1 [43"].

Oct The octamer proteins also regulate the IL-2 promoter by binding to two octamer-binding sites within it ([44]; see Fig. 2). Mutation of either of these sites results in a partial

loss of IL-2 promoter activity, but mutation of both sites abolishes promoter function [22,44]. An inducible site hypersensitive to DNAse I maps just upstream of the proximal, higher affinity site [6] and in vivo footprinting confirms that this site is occupied in stimulated cells [7°'].

Like the distal NFAT site, the proximal Oct site is a composite element, which that supports the cooperative binding of Oct and AP-1 proteins ([45,46"',47°°,48°]; see Fig. 2). Mutations that simultaneously disrupt both the Oct site and the AP-1 site within the proximal Oct element eliminate IL-2 promoter function, even though the distal Oct site is unchanged [45,47"']. Both Oct-1 and Oct-2 proteins bind to the proximal Oct site and cooperate functionally with Fos- and Jun-family proteins [44,46"',47"',48"]. Although Oct-2 is clearly superior to Oct-1 as a transactivator at this site [44,47°%48"]. Oct-1 and Oct-2 differ in their expression patterns (Fig. 1): whereas Oct-1 is constitutively expressed, Oct-2 is newly synthesized in cells activated with antigen [49]. The identities of the optimal Fos and Jun partners for Oct-1 and Oct-2 vary in different reports [46..,47.',48"].

Despite its striking activity in transient transfection assays, Oct-2 is not essential for IL-2 gene transcription. Mice deficient in Oct-2 show no gross abnormalities in peripheral T-cell function [50]. Moreover, Jurkat T cells, which are reported to lack Oct-2 [44], are clearly capable of IL-2 gene transcription. Other Oct-family proteins (Oct-T1, Oct-T2, and Oct-T3) that bind to the proximal Oct site have been described, and may influence IL-2 gene transcription [51].

NFKB Mutations in the NFKB site of the IL-2 promoter are in general less deleterious than mutations at other sites (reviewed in [13"]). Nevertheless, in vivo footprinting of the IL-2 promoter indicates that the expected bases of the NFIc.B site are contacted by a nuclear factor


in stimulated (but not unstimulated) T cells ([7°°]; see Fig. 2).

Although most Rel-family proteins are present in T cells, the major nuclear factor binding to the IL-2 NF~d3 site shortly after stimulation is probably the p50-p65 (NFrd31-RelA) heterodimer ([52"',53,54"; see [55] for NFrd3 nomenclature; Fig. 1). In response to a variety of extracellular stimuli, p50-p65 NFrd3 is rapidly released from an inactive cytoplasmic pool by the rapid phosphorylation and subsequent degradation of the inhibitor Ird3ct [56°'], or by a less well understood mechanism involving the related inhibitor Ird3~ [57"']. Release unmasks the nuclear localization signal of the p50-p65 heterodimer, and leads to its translocation into the nucleus (reviewed in [52°',55]). The proteolytic processing of precursor proteins to yield p50 and p52 (NFrd32) is also highly regulated [58]. Finally, phosphorylation of NF~cJ3 proteins may be required for fully functional activity [59,60].

At later times after activation, c-Rel may be a significant component of the complexes binding to the IL-2 NFrd3 site (Fig. 1). The levels of total and nuclear c-Rel in T cells increase considerably following stimulation [61°,62°]. As c-Rel-p65 heterodimers are strongly transactivating in other systems [63], c-Rel may be more likely to maintain rather than to repress the late transcription of IL-2 mRNA. RelB, which does not homodimerize but which forms heterodimers with p50 or p52, is constitutively expressed in the nucleus of lymphoid cells [64,65]. Mice deficient in p50 or RelB, however, do not show significant impairment of T-cell function [66,67]. The function of p52 (NFK.B2) in IL-2 transcription has not been investigated.

The p50 homodimer may serve an important regulatory function, especially in untransformed T cells [53,54"]. Dimers of p50, which do not possess a transactivation domain and often repress transcription [64], are constitutively present in nuclear extracts of several T-cell clones [53,54°]. Activation of these cells results in a decrease in the level of p50 dimers in the nucleus and a concomitant increase in the level of active p50-p65 heterodimers [53,54"], by a mechanism potentially involving the inhibitor Bcl-3 [68]. As the constitutive presence of nuclear p50 dimers was not noted in other studies with untransformed T cells [69,70], it may be that p50 dimers repress IL-2 gene transcription in specific T-cell subsets (e.g. T helper type 2 cells; [54"]), or in defined states of anergy or tolerance.

AP-1 (Fos-Jun) The AP-1 site of the IL-2 promoter is occupied in stimulated EL4 T cells, although the in vivo footprint is not entirely consistent with that predicted from studies -of AP-1 binding in vitro [7"*]. Mutation of this site abolishes promoter function in transient transfection assays [71]; in vitro, this site binds dimers of Fos- and Jun-family proteins (Fos,.FosB, Fra-1, Fra-2 and Jun, JunB and JunD, respectively [16,71,72,73",74]). The

dimerization affinities of Fos-Jun heterodimers are much higher than those ofJun-Jun homodimers [74]; hence, Fos-Jun dimers are also more effective at DNA binding and transactivation. Because the AP-1 site of the IL-2 promoter is a relatively low-affinity site [16,71], it may require Fos-Jun dimers for optimal activity; thus, the requirement for protein synthesis for IL-2 gene induction [4] may reflect, in part, a requirement for de novo synthesis of Fos-family proteins ([71,75]; see Fig. 1). Constitutive overexpression of c-Fos in transgenic mice results in increased production of IL-2 m R N A and protein by stimulated T cells [76]. Neither c-Fos nor c-Jun provides an irreplaceable function in IL-2 gene transcription, as judged by analysis of mutant mice [39,77].

Induced soon after stimulation (Fig. 1), c-Fos and c-Jun are potent transactivators, whereas Fra-1 and Fra-2, which appear later, lack transactivation domains [78]. Thus, replacement of c-Fos and FosB with Fra-1 and Fra-2 is likely to attenuate IL-2 transcription. AmongJun-family proteins, c-Jun andJunB are generally transactivating [79], but JunB is inefficient in comparison with c-Jun and can repress transactivation mediated by c-Jun if overexpressed [80]. JunD is a negative regulator of proliferation and transformation in fibroblasts [81]; its function in IL-2 gene transcription is unknown.

Post-translational modification of Fos and Jun proteins is also likely to influence their role in IL-2 gene transcription. A serine/threonine kinase that phos- phorylates a carboxy-terminal peptide of c-Fos in vitro is rapidly activated in T cells stimulated with anti-CD3 [82]. Jun amino-terminal kinases (INKs), which belong to the greater family ofmitogen-activated protein (MAP) kinases (reviewed in ~83"]), activate c-Jun by phosphorylating two serine residues in its amino-terminal activation domain [84"']; the relevant isozyme is likely to be JNK2 [85]. An interesting point is that JNK activity is differently regulated in T cells and non-T cells. In T cells, combined stimulation with phorbol ester and calcium ionophore is required for maximal JNK activation [84"*], and consequently for maximal transactivation mediated by AP-1 [37",86"']; stimulation with phorbol ester alone is sufficient for this activation in non-T cells [84"]. The requirement for dual stimulation is especially apparent in primary T cells [86"'], or when single AP-.1 sites [84"'] or multimers of weak sites (such as the IL-2 promoter AP-1 site [37"]) are used.

A particularly striking aspect oflL-2 gene transcription is the participation of AP-1 proteins at multiple regulatory sites (NFAT, Oct, and AP-1 itself). Cooperation with transcription factors such as NFATp may specify the orientation of Fos-Jun dimers on adjacent sites ([73"]; L Chen, GL Verdine, personal communication), thus conferring a precise architecture on the entire promoter/enhancer complex. Curiously, the use of a dominant-negative Jun suggests certain differences in the properties of these three complexes containing AP-1 [37"]. This dominant-negative protein (which lacks the

Transcriptional regulation of the IL-2 gene Jain, Loh and Rao 337

transactivation domain but contains the DNA-binding domain of c-Jun) does not downregnlate transcription from the IL-2 promoter AP-1 site or the proximal Oct site, although it strongly inhibits transcription from the distal NFAT site or a consensus AP-1 site [37"]. The reason for this difference is not clear: one possibility is that the IL-2 AP-1 and Oct elements are weaker AP-1 sites that can bind only Fos-Jun dimers, and hence the dominant-negative Jun would function to recruit transactivating Fos proteins to the sites.

Cooperative interactions among transcription factors

There is a growing awareness that gene transcription is not merely the sum of multiple independent interactions of transcription factors with the basal transcription complex. Rather, transcription is governed by the formation of highly cooperative assemblies containing a precise arrangement of many proteins, including transcription factors, DNA-binding proteins that serve an architectural function, and coactivators that bridge the transcription factors and the basal transcription complex [87",88"'].

The results of in vivo footprinting provide good evidence that such interdependent transcription complexes form on the IL-2 promoter. Although the TATA box may be constitutively occupied [89], there is no detectable occupancy of other regulatory elements of the IL-2 promoter in unstimulated T cells [7"']. In contrast, coordinate occupancy of all sites is observed as early as one hour following stimulation [7"]. Inhibitors (CsA, forskolin) which decrease IL-2 mRNA induction cause a global loss of the in vivo footprint, despite the fact that each compound interferes with a distinct signal transduction pathway and inhibits the DNA binding of only a subset of factors in vitro [7",90]. Thus, stable occupancy of the promoter may require simultaneous binding to all sites, and interference with the binding of even a single component may abolish formation of the entire enhancer complex.

The individual regulatory elements in the IL-2 promoter are non-consensus sites that bind their cognate factors with three to tenfold lower aflqnity than the corresponding consensus sites [91]. This arrangement appears to be necessary for maintaining the cell specificity of IL-2 promoter expression, as mutation of each of the non-consensus elements to the corresponding consensus sites unexpectedly results in IL-2 promoter expression even in non-T cells [91]. Again, cooperative interactions between factors binding to weak sites may be required to minimize inappropriate gene expression, and to render the entire complex more responsive to developmental and extracellular stimuli.

Calcineurin

Modification of the phosphorylation status of transcription factors is a key mechanism for regulating their

function [92",93]. The calcium-dependent phosphatase calcineurin plays a major role in this process in T cells and other cells involved in the immune response. The involvement of calcineurin has been established through the use of the immunosuppressive drugs CsA and FK506, which inhibit calcineurin activity when it forms a complex with specific immunophilin receptors, ([94,95]; reviewed in [13",96,97]).

When added before the stimulus, CsA and FK506 inhibit the induced transcription of IL-2 and other cytokine genes [3,4], indicating that calcineurin is required to initiate gene transcription. CsA also inhibits ongoing IL-2 transcription, indicating that there is a continuing requirement for active calcineurin during IL-2 transcription [4,7",8,9"']. One known target of calcineurin is the pre-existing, cytoplasmic component of NFAT [32"',98,23"']; other targets may be JNK-family kinases and IKBct ([84"',99"']; see below). CsA and FK506 also completely or partially inhibit the induction of mRNAs encoding several nuclear factors implicated in IL-2 transcription (NFATc, Oct-2, pl05/NFKB1; [24",49,100]). Thus calcineurin, acting in concert with other signal transduction pathways, may regulate IL-2 gene transcription pardy by activating pre-existing transcription factors, and partly by modulating the synthesis of transcription factors that are required for optimal assembly of the cooperative enhancer complex.

NFAT NFAT-family proteins are a primary target of calcineurin. Calcineurin mediates a rapid, site-specific dephosphorylation of NFATp in activated T cells (KTY Shaw, A Ho, A Rao, PG Hogan, personal communication), either directly or by activating a phosphatase cascade. The dephosphorylation precedes the translocation of NFATp into the nucleus and is associated with an increase in its affinity for DNA (A Ho, J Kim, J Jain, A P, ao, PG Hogan, personal communication). Upon withdrawal of the simulus, or upon addition of CsA in the continuing presence of stimulus, NFATp is rapidly rephosphorylated and again localizes to the cytoplasm (C Loh, A P, ao, unpublished data). This phenomenon may underlie the ability of CsA to inhibit ongoing IL-2 transcription [4,7"',9"'].

Calcineurin is also likely to modulate transactivation mediated by the NFAT-Fos-Jun complex by virtue of its effect on AP-1 (see below); whether it affects the intrinsic transcriptional activities of NFAT-family proteins has not yet been determined. Overexpression of calmodulin-dependent kinases has been reported to inhibit NFAT-dependent transactivation [101",102"]. These kinases may reverse or prevent the dephosphorylation of NFAT-family proteins, or cause their phosphorylation at inappropriate sites.

Oct Induction of the Oct/AP-1 DNA-binding complex is unaffected by CsA and FK506, but transactivation driven


by multimers of the proximal Oct site is highly sensitive to these drugs [46"°,47°°]. Again there are at least two potential mechanisms for this effect: the calcineurin dependence of AP-1 function in T cells (see below), and the sensitivity of Oct-2 synthesis to CsA [49].

NFKB/Rel Calcineurin is likely to increase NFKB activity by several mechanisms, as inferred from the inhibitory effects of CsA and FK506. An immediate effect of calcineurin may be to potentiate (indirectly) the phosphorylation and degradation of IKB(x, thus leading to an increase in the level of active nuclear NFKB [99"]. A late effect of calcineurin, occurring several hours after initial stimulation, is to increase the level of nuclear c-Rel [61°], by increasing c-Rel synthesis and/or potentiating its nuclear translocation. Calcineurin is also involved in augmenting the induction of pl05/NFKB1 mRNA, especially in primary T cells [100], and in decreasing the level of p50 dimers in the nucleus of T cells activated with antigen [53]. The combined result of these calcineurin-mediated processes would be to increase the effective ratio o f activating to inactive complexes and thus to potentiate transactivation from the NFKB site [53,54"].

AP-1 As mentioned above, combined stimulation with phorbol ester and calcium ionophore is required for maximal activation of JNK and maximal AP-1 mediated transactivation in T cells [37°,84"°,86"°]. Both effects are blocked by CsA and FK506, implicating calcineurin in a T cell specific pathway of JNK activation and consequent activation ofc-Jun [84°',86°']. The mechanism by which calcineurin regulates JNK activity in T cells remains to be understood.

Costimulatory pathways that augment IL-2 production

Optimal cytokine production, especially by primary T cells, requires the engagement of costimulatory receptors and the TCK. Although additional costimulatory pathways exist [103,104], we focus here on CD28, a receptor that interacts with B7-family proteins expressed on antigen-presenting cells (reviewed in [105",106°°]).

The mechanism and magnitude of CD28-mediated costimulation has been carefully assessed in a murine T-cell clone [9°°]. Combined stimulation with anti-CD28 and anti-TCR antibodies resuked in a 30 to 100-fold increase in IL-2 accumulation over that observed with ant i-TCR stimulation alone. Quantitation of IL-2 m R N A levels indicated that CD28 exerted both nuclear and post-nuclear effects. The nuclear effect was inferred from the eightfold increase in the levels ofunspliced (i.e. newly transcribed) IL-2 m R N A in cells costimulated with anti-CD28. This was reflected in a 20-fold increase in the levels of cytoplasmic IL-2 mRNA, even at the earliest times at which ~IL-2 m R N A was detectable by

polymerase chain reaction analysis. The post-nuclear effects were even more striking: CD28 costimulation caused a pronounced increase in the stability of IL-2 m R N A [9"°,10], and hence delayed the kinetics of its disappearance by several hours [9"°].

It is unclear whether the nuclear effect of CD28 costimulation is exerted at the transcriptional level (reviewed in [106"°,107]). In cells stably transfected with IL-2 promoter plasmids, CD28 costimulation had no effect on luciferase expression driven by up to 1.9 kb of the IL-2 promoter [9"'], suggesting that transcription initiation was not involved. In other systems, however, CD28 costimulation augments IL-2 promoter activity by three to fivefold [106°°,107-109]. This effect has been mapped to a CD28 response element located just 5' of the AP-1 site (see Fig. 2).

At least two transcription factors, NFKB/Rel and AP-1, may be targets for CD28 costimulation [62",84"°,86°°]. The CD28 response element is reported to bind tkel- family proteins, and CD28 signalling results in sustained downregulation of IKB~ and increased translocation of c-Rel into the nucleus [62"]. Moreover, purified T cells from mice deficient in p50 NFKB show decreased responses to CD28 costimulation [66]. CD28 costimulation also enhances transactivation mediated by multimers of a consensus AP-1 site in transgenic mice [84°',86°°]; the mechanism may involve a CD28-mediated increase in the activity ofJNK-family kinases [84°°].

Conclusions

As a strongly synergistic promoter with complex kinetics of activation, the IL-2 promoter provides a valuable paradigm for studies of transcriptional regulation. IL-2 gene transcription reflects the convergence of multiple signal transduction pathways on at least four unrelated families of transcription factors, whose functions are controlled at the level ofposttranslational modification as well as at the level of de novo synthesis. The steady-state level of IL-2 m R N A that is achieved is further regulated by a poorly understood inducible mechanism for degradation of cytokine mRNAs, which appears to be inhibited by costimulation through CD28. Together these processes provide a mechanism for regulated and continuous control of IL-2 gene expression in specific lineages of immune system eells.

Note added in proof

The study referred to in the text as T Hoey, S Ya-Lin, K Williamson, X Xu, personal communication, has now been published [111]. The reader is also referred to a recent, more detailed, review on IL-2 gene transcription [112].

Acknowledgements

We thank Scott Umlauf, Ron Schwartz, Naoko Arai, Tim Hoey, Sankar Ghosh, Edgar Serfling, Tom Curran, Lin Chen,

Transcr ip t ional regulat ion o f the IL-2 gene Jain, Loh and Rao 339

Greg Verdine, Patrick Hogan, and Laurie Glimcher for mak- ing their data available prior to publication; Scott Umlauf, Ellen Rothenberg, Barbara Wold, Ranjan Sen and Andy Lichtman for discussions; Patrick Hogan for critical reading of the manuscript; Chris Gallegos for assistance with preparation of the manuscript; and the members of the laboratory for their contributions to this work. We apologize to those colleagues whose work we have been unable to cite because of space, limitations. This work was supported by NIH grants CA42471, GM42667 and A135297 (to A Rao) and a Barr Program Small Grant (to J Jain). J Jain is a Special Fellow and A Rao a Scholar of the Leukemia Society of America. C Loh is a Ryan Foundation Fellow.

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clones. Both nuclear and cytoplasmic RNA are regulated with complex kinetics. Mol Cell Biol 1995, 15:3197-3205.

A quantitative analysis of the effect of CD28 on IL-2 transcription. The authors show that CD28 costimulation delays the degradation of IL-2 mRNA and also has a nuclear effect that may not involve transcription.

10. Lindsten T, June CH, Ledbetter JA, Stella G, Thompson CB: Regulation of lymphokine messenger RNA stability by a surface-mediated T-cell activation pathway. Science 1989, 244:339-343.

11. Bohjanen PR, Petryniak B, June CH, Thompson CB, Lindsten 1: An inducible cytoplasmic factor (AU-B) binds selectively to AUUUA multimers in the 3' Untranslated region of lymphokine mRNA. Mol Cell Biol 1991, 11:3288-3295.

12. Nair APK, Hahn S, Banhoizer R, I-lirsch HH, Moroni C: Cydosporin A inhibits growth of autocrine tumour cell lines by destabilizing interleukin-3 mRNA. Nature 1994, 369:239-242.

13. Crabtree GR, Clipstone NA: Signal transmission between the • plasma membrane and nucleus of T-lymphocytes. Annu Rev

Biochem 1994, 63:1045-1083. A recent review of the events of T-cell activation that lead to inducible gene transcription.

14. Novak TJ, White PM, Rothenberg EV: Regulatory anatomy of the murine Interleukln-2 gene. Nucleic Acids Res 1990, 18:4523-4533.

15. Durand DB, Shaw JP, Bush MR, Replogle RE, Belagaje R, Crabtree GR: Characterization of antigen receptor response elements within the intedeukin-2 enhancer. Mol Cell Biol 1988, 8:1715-1724.

16. Serfling, E, Barthelmas P, Schenk B, Zarius S, Swoboda R, Mercurio F, Karin M: Ubiquitous and lymphocyte-specific factors are involved in the induction of the mouse interleukin 2 gene in T-lymphocytes. EMBO J 1989, 8:465-473.

17. Brombacher F, Schafer T, Weissenstein U, Tschopp C, Andersen • E, Burki K, Baumann G: IL-2 promoter-driven lacZ expression

as a monitoring tool for IL-2 expression in primary T-cells of transgenlc mice. Int Immunol 1994, 6:189-197.

The first description of transgenic mice bearing an IL-2 promoter-reporter transgene. Only one of 17 founder lines showed inducible, T cell specific expression of the lacZ reporter gene.

18. Palmiter RD, Sandgren EP, Koeller DM, Brinster RL: Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mice. Mol Cell giol 1993, 13:5266-5275.

19. Nolan GP: NF-AT-1 and ReI-bzIP: hybrid vigor and binding under the influence. Cell 1994, 77:795-798.

20. Rao, A: NF-ATp: a transcription factor required fur the • • coordinate induction of several cytokiue genes. Immunol Today

1994, 15:274-281. A review of the role of nuclear factor of activated T cells (NFAT) proteins in inducible, cyclosporin A-sensitive cytokine gene transcription, focus- ing on their cooperative interactions with Fos- and Jun-family proteins.

21. Thompson CB, WangC-Y, Ho I-C, Bohjanen PR, Petryniak B, June CH, Miesfeldt S, Zhang L, Nabel CJ, Karpinski B, Leiden JM: c/s-acting sequences required for inducible interleukin-2 enhancer function bind a novel Ets-related protein, EIf-l. Mol Cell Biol 1992, 12:1043-1053.

22. Zhang L, Nabel GJ: Positive and negative regulation of IL-2 gene expression: role of multiple regulatory sites. Cytokine 1994, 6:221-228.

23. McCaffrey PG, Perrino BA, Soderling TR, Rao A: NF-ATp, °• a T-lymphocyte DNA-binding protein that is a target for

calcineurio and immunosuppressive drags. ] Biol Chem 1993, 268:3747-3752.

See annotation {25•e].

24. Northrop JP, Ho SN, Chen L, Thomas DJ, Timmerman LA, Nolan • e GP, Admon A, Crabtree GR: NF-AT components define a family

of transcHptlon factors targeted in T-cell activation. Nature 1994, 369:497-502.

See annotation [25••].

25. Masuda ES, Naito Y, Tokumitsu H, Campbell D, Saito F, • • Hannum C, Arai K, Arai N: NFATx: a novel member of the

NFAT family that is predominantly expressed in the thymus. Mol Cell giol 1995, 15:2697-2706.

Along with [23ee,24••1, this paper describes the cloning and characterization of cDNAs encoding three distinct nuclear factor of activated T cells (NFAT)-family proteins.

26. Wang DZ, McCaffrey PG, Rao A: The cyciosporin-sensitive transcription factor NFATp is expressed in several classes of cells in the immune system. Ann NY Acad Sci 1995, 776:in press.

27. Castigli E, Pahwa R, Good RA, Geha RS, Chatila TA: Molecular basis of a multiple lymphokine deficiency in a patient with severe combined immunodeflciency. Proc Natl Acad Sci USA 1993, 90:4728-4732.

28. lain J, Burgeon E, Badalian TM, Hogan PG, Rao A: A similar • • DNA-binding motif in NFAT family proteins and the Rel

homology region. J Biol Chem 1995, 270:4138-4145.

3 4 0 Lymphocyte ac t iva t ion and ef fector funct ions

The minimal DNA-binding domain of NFATp contains a sequence motif that is structurally and functionally related to the DNA-recognition loop of NFKB/Rel proteins.

29. Ghosh G, Duyne GV, Ghosh S, Sigler PB: Structure of • NF-kB p50 homndimer bound to a kB site. Nature 1995,

373:303-310. See annotation [30•1.

30. Muller CW, Rey FA, Sodeoka M, Verdine GL, Harrison SC: • Structure of the NF-KB p50 homodimer bound to DNA. Nature

1995, 373:311-317. This paper and [29"] report the crystal structure of the Rei-homology region of p50 homodimers bound to DNA.

31. McCaffrey PG, Goldfeld AE, Rao A: The role of NFATp in cyclnsporin A-sensitive tumor necrosis factor<x gene transcription. ] Biol Chem 1994, 269:30445-30450.

32. Jain J, McCaffrey PG, Miner Z, Kerppola TK, Lambert JN, "" Verdine GL, Curran 1, Rao A: The T-ceil transcription factor

NFATp is a substrate fur calcinenrin and interacts with Fos and Jun. Nature 1993, 365:352-355.

The minimal DNA-binding domains of Fos and Jun are sufficient for cooperative interaction with NFATp on the IL-2 promoter NFAT site.

33. Yaseen NR, Park J, Kerppola 1, Curran T, Sharma S: A central role fur Fos in human B- and T-cell NFAT (nuclear factor of activated T-ceils): an acidic region is required for in vitro assembly. Mol Cell Biol 1994, 14:6886-6895.

34. Jain J, McCaffrey PG, Valge-Archer VE, Rao A: Nuclear factor of activated T-ceils contains Fos and Jon. Nature 1992, 356:801-804.

35. Northrop JP, UIIman KS, Crabtree GR: Characterization of the nuclear and cytoplasmic components of the lymphold-specific nuclear factor of activated T-ceils (NF-AT) complex. J Biol Chem 1993, 268:2917-2923.

36. Rooney JW, Hoey T, Glimcher LH: Coordinate and cooperative • " roles fur NF-AT and AP-1 in the regulation of the murine IL-4

gene. Immunity 1995, 2:473-483. Analysis of the cooperative interactions of nuclear factor of activated T cells (NFAT)-family proteins with Fos and Jun-family proteins on the IL-4 promoter NFAT site; emphasizes the similarity to the IL-2 promoter NFAT site.

37. Petrak D, Memon SA, Birrer MJ, Ashwell )D, Zacharchuk • CM: Dominant negative mutant of c-Jun inhibits NF-AT

transcriptional activity and prevents IL-2 gene transcription. J Immunol 1994, 153:2046-2051.

Distinct effects of a dominant-negative Jun on transactivation from the IL-2 promoter site and from several muhimerized regulatory elements.

38. Boise LH, Petryniak B, Mao X, June CH, Wang C-Y, Lindsten T, Bravo R, Kovary K, Leiden JM, Thompson CB: The NFAT-1 DNA binding complex in activated T-cells contains Fra-1 and JunB. Mol Cell Biol 1993, 13:1911-1919.

39. Jain J, Nalefski EA, McCaffrey PG, Johnson RS, Spiegelman BM, Papaioannou V, Rao A: Normal peripheral T-ceU function in cFos-deficient mice. Mol Cell Biol 1994, 14:1566-1574.

40. Chuvpilo S, Schomberg C, Gerwig R, Heinfling A, Reeves R, Grummt F, Serfling E: Multiple closely-linked NFAT]Octamer and HMG I(Y) binding sites are part of the intedeukin-4 promoter. Nucleic Acids Res 1993, 21:5694-5704.

41. Li C, Lai C, Sigman DS, Gaynor RB: Cloning of a cellular factor, Interleukin Binding Factor, that binds to NFAT-Ilke motifs in the human immunndeficlency virus long terminal repeat. Proc Natl Acad Sci USA 1991, 88:7739-7743.

42. Kao PN, Chen L, Brock G, Ng J, Kenny J, Smith AJ, Corthesy B: Cloning and expression of cyclosporin A- and FKS06-sensitive nuclear factor of activated T-cells: NF45 and NF90. J giol Chem 1994, 269:20691-20699.

43. Romano-Spica V, Georgiou P, Suzuki H, Papas TS, Bhat NK: • " Role of ETS1 in IL-2 gene expression. J Immunol 1995,

154:2724-2732. Jurkat cells stably overexpressing Ets-1 show a marked decrease in IL-2 production, whereas Jurkat cells overexpressing antisense Ets-1 show an increase in IL-2 production and Ib-2 promoter activity.

44. Kamps MP, Corcoran L, Lebowitz JH, Baltimore D: The promoter of the human Interleukin-2 gene contains two octamer.binding sites and is partially activated by the expression of Oct-2. Mol Cell Biol 1990, 10:5464-5472.

45. UIIman KS, Flanagan WM, Edwards CA, Crabtree GR: Activation of early gene expression in T-lymphocytes by Oct-1 and an inducible protein, OAP 40. Science 1991, 254:558-562.

46. UIIman KS, Northrop JP, Admon A, Crabtree GR: Jun family *" members are controlled by a calcium-regulated, cyclosporin A-

sensitive signaling pathway in activated T-lymphocytes. Genes Dev 1993, 7:188-196.

See annotation [48"1.

47. Pfeuffer I, Klein-Hessling S, Heinfling A, Chuvpilo S, Escher C, .e Brabletz 3, Hentsch B, Schwarzenback H, Mathias P, Serfling

E: Octamer factors exert a dual effect on the IL-2 and It-4 promoters. J Immunol 1994, 153:5572-5585.

See annotation [48"].

48. De Grazia U, Felli MP, Vacca A, Farina AR, Maroder M, • Cappabianca L, Meco D, Farina M, Screpanti I, Frail L,

Gulino A: Positive and negative regulation of the composite octamer motif of the intedeukin 2 enhancer by AP-1, Oct-2 and retinoic acid receptor. J Exp Med 1994, 180:1485-1497.

Along with [46••,47"], this paper demonstrates cooperation between proteins of the Oct and activating protein-1 families on the proximal octamer site of the IL-2 promoter, and shows that transactivation from the site is sensitive to cyclosporin A.

49. Kang S-M, Tsang W, Doll S, Schede P, Ko, H-S, Tran A-C, Lenardo MJ, Staudt LM: Induction of the POU doolain transcription factor Oct-2 during T-ceil activation by cognate antigen. Mol Cell Biol 1992, 12:3149-3154.

50. Corcoran LM, Karvelas M: Oct-2 is required early in T.ceil independent B-cell activation fur G1 progression and for proliferation. Immunity 1994, 1:635-645.

51. Bhargava AK, Li A, Weissman SM: Differential expression of four members of the POU family of proteins in activated and phorbol 12-myrlstate 13-acetate-treated Jurkat T-cells. Proc Natl Acad Sci USA 1993, 90:10260-10265.

52. Thanos D, Maniatis T: NF-KB: A lesson in family values. Cell • • 1995, 80:529-532. A highly recommended review on the regulation and function of N FKB/Rel proteins.

53. Kang S-M, Tran A-C, Grilli M, Lenardo MJ: NF-KB subunit regulation in nontransformed CD4 ÷ T.Iymphoo/tes. Science 1992, 256:1452-1456.

54. Lederer JA, Liou JS, Todd MD, Glimcher LH, Lichtman AH: • Regulation of cytokine gene expression in T-helper cell subsets.

J Immunol 1994, 152:77-86. Along with [53], this paper suggests that homodimers of p50 NFKB inhibit IL-2 gene transcription whereas heterodimers of p50/p65 NFKB activate it.

55. Siebenlist U, Franzoso G, Brown K: Structure, regulation and function of NF-KB. Annu Rev Cell Biol 1994, 10:405-455.

56. Brown K, Gerstberger S, Carlson L, Franzoso G, Siehenlist U: • • Control of IKB-a proteolysis by site-specific, signal-induced

phosphorylation. Science 1995, 267:1485-1488. See annotation [57"'].

57. Thompson JE, Phillips RJ, Erdjument-Bromage H, Tempst P, "" Ghosh S: IKB-i3 regulates the persistent response in a biphasic

activation of NF-r,B. Cell 1995, 80:573-582. This paper and [56 •] are stellar papers on the regulation of NFKB by IKBcc and IKB~3.

58. Palombella VJ, Rando OJ, Goldberg AL, Maniatis T: The ubiquitin-proteasome pathway is required for processing the NF-KB1 precursor protein and the activation of NF-KB. Cell 1994, 78:773-785.

59. Li C-CH, Dai R-M, Chen E, Longo DL: Phosphorylation of NF-KBI-p50 is involved in NF.KB activation and stable DNA binding. J Biol Chem 1994, 269:30089-30092.

60. Naumann M, Scheidereit C: Activation of NF-KB in vivo is regulated by multiple phnsphoryfations. EMBO J 1994, 13:4597-4607.

Transcr ipt ional regulat ion o f the IL-2 gene Jain, Loh and Rao 341

61. Lakshmi V, Burakoff SJ, Sen R: FK506 inhibits antigen receptor- . mediated induction of c-Rel in B- and T-lymphoid cells. J Exp

Med 1995, 181:1091-1099. See annotation [62e].

62. Bryan RG, I.i Y, Lai J-H, Van M, Rice NR, Rich RR, Tan • T-H: Effect of CD28 signal transduction on c-Rel in human

peripheral blood T-cells. Mo/ Cell gio/ 1994, 14:7933-7942. Along with [61"], this paper shows that c-Rel translocates to the nucleus of activated T cells, and is regulated by two independent signalling pathways involving calcineurin and CD28 costimulation.

63. Hansen SK, Baeuerle PA, Blasi F: Purification, reconstitution, and Ir, B association of the c-Rel-p65 (relA) complex, a strong activator of transcription. Mo/ Cell Biol 1994, 14:2593-2603.

64. Dobrzanski P, Ryseck R-P, Bravo R: Differential interactions of ReI-NF-r..B complexes with IKBa determine pools of constitutive and inducible NF-KB activity. EMBO J 1994, 13:4608-4616.

65. Lerobecher T, Kistler B, Wirth T: Two distinct mechanisms contribute to the constitutive activation of RelB in lymphoid cells. EMBO J 1994, 13:4060-4069.

66. Sha WC, Liou H-C, Tuomanen El, Baltimore D: Targeted disruption of the p50 subonit of NF-tcB leads to multifocal defects in immune responses. Cell 1995, 80:321-330.

67. Weih F, Carrasco D, Durham SK, Barton DS, Rizzo CA, Ryseck R-P, Lira SA, Bravo R: Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-KB/Rel family. Cell 1995, 80:331-340.

68. Franzoso G, Bours V, Azarenko V, Park S, Tomita-Tamaguchi M, Kanno T, Brown K, Siebenlist U: The oncoprotein Bcl-3 can facilitate NF-KB-mediated transactivation by removing inhibiting I)50 homndimers from select KB sites. EMBO ] 1993, 12:3893-3901.

69. Jamieson C, McCaffrey PG, Rao A, Sen R: Physiologic activation of T-cells via the T.cell receptor induces NF-KB. ] Immunol 1991, 147:416-420.

70. Jain J, Valge-Archer VE, Sinskey AJ, Rao A: The AP-1 site at -1501)1), but not the NF-KB site, is likely to represent the major target of protein kinase C in the intedeukin 2 promoter. J Exp Med 1992, 175:853-862.

71. Jain J, Valge-Archer VE, Rao A: Analysis of the AP-1 sites in the IL-2 promoter. J Immunol 1992, 148:1240-1250.

72. Kerppola TK, Curran 1.' Transcription factor interactions: basics on zippers. Curr Opin Struct Biol 1991, 1:71-79.

73. Glover JNM, Harrison SC: Crystal structure of the her- • erodimeric bZlP transcription factor c-Fos-c-Jun bound to

DNA. Nature 1995, 373:257-261. This paper reports the crystal structure of a Fos-Jun dimer bound to DNA, thus illuminating the structural basis for homodimerization and heterodimerization of bZIP-family proteins.

74. Ryseck R-P, Bravo R: c-Jun, JunB, and JunD differ in their binding affinities to AP-1 and CRE consensus sequences: effect of Fos proteins. Oncogene 1991, 6:533-542.

75. Kovary K, Bravo R: Expression of different Jun and Fos proteins during the G0-to-G 1 transition in mouse fibroblasts: in vitro and in vivo associations. Mol Cell Bio/ 1991, 11:2451-2459.

76. Ochi Y, Koizumi T, Kobayashi S, Phuchareon J, Hatano M, Takada M, Tomita Y, Tokuhisa 1-." Analysis of IL-2 gene regulation in c-Fos transgenic mice. ] Immunol 1994, 153:3485-3490.

77. Chen J, Stewart V, Spyrou G, Hilberg F, Wagner EF, AIt FW: Generation of normal T- and B-lymphocytes by c-jun deficient embryonic stem cells. Immunily 1994, 1:65-72.

78. Wisdom R, Verma IM: Transformation by Fos proteins requires a C-terminal transactivation domain. Mol Cell Biol 1993, 13:7429-7438.

79. Suzuki T, Okuno H, Yoshida T, Endo T, Nishina H, Iba H: Difference in transcriptional regulatory function between c-Fos and Fra-2. Nucleic Acids Res 1991, 19:5537-5542.

80. Chiu R, Angel P, Karin M: JunB differs in its biological properties from, and is a negative regulator of, cJun. Cell 1989, 59:979-986.

81. Pfarr CM, Mechta F, Spyrou G, Lallemand D, Carillo S, Yaniv M: Mouse JunD negatively regulates fibroblast growth and antagonizes transformation by ras. Cell 1994, 76:747-760.

82. Nel AE, Taylor LK, Kumar GP, Gupta S, Wang SC-T, Williams K, Liao O, Swanson K, Landreth GE: Activation of a novel serine/threonine kinase that phnsphorylates c-Fos upon stimulation of T- and B-lymphocyles via antigen and cytokine receptors. J Immunol 1994, 152:4347-4357.

83. Davis RJ: MAPKs: new JNK expands the group. Trends Biochem e. Sci 1994, 19:470-473. A pertinent review on the extended mitogen-activated protein (MAP) kinase family.

84. Su B, ]acinto E, Hibi M, Kallunki T, Karin M, Ben-Neriah Y: " JNK is involved in signal integration during costimulation of

T-lymphocytes. Cell 1994, 77:727-736. Activation of Jun amino-terminal kinases (JNKs) in T cells but not in fibroblasts requires calcineurin activity. JNK activation is also augmented by CD2B costimulation.

85. Sluss HK, Barrett T, Derijard B, Davis RJ: Signal transduction by tumor necrosis factor mediated by JNK protein kinases. Mol Cell Biol 1994, 14:8376-8384.

86. Rincon M, Flavell RA: AP-1 transcription activity requires both • " T-cell receptor-mediated and costimulatory signals in primary

T-lymphocyles. EMBO J 1994, 13:4370-4381. Transactivation mediated by a consensus activating protein-1 (AP-1) site in T cells of transgenic mice is dependent on calcineurin and CD2B costimulation, although AP-1 binding activity can be elicited by stimulation with phorbol ester alone.

87. Tjian R, Maniatis T: Transcriptional activation: a complex " puzzle with few easy pieces. Cell 1994, 77:5-8. A thought-provoking review of transcriptional regulation mediated by 'stereospecific enhancer complexes' interacting with the basal transcription complex.

88. Robertson LM, Kerppola TK, Vendrell M, Luk D, Smeyne ee RJ, Bocchiaro C, Morgan Jl, Curran T: Regulation of c-Fos

expression in tramgenic mice requires multiple interdependent transcription control elements. Neuron 1995, 14:241-252.

An experimental illustration of the concepts reviewed in [87ee], using the c-Fos promoter in transgenic mice.

89. Brunvand MW, Krumm A, Groudine M: In vivo footprinting of the human IL-2 gene reveals a nuclear factor bound to the transcription start site in T-cells. Nucleic Acids Res 1993, 21:4824-4829.

90. Chen D, Rothenberg EV: Interleukin 2 transcription factors as molecular targets of cAMP inhibition: delayed inhibition kinetics and combinatorial transcription roles. J Exp Med 1994, 179:931-942.

91. Hentsch B, Mousaki A, Pfeuffer I, Rungger D, Serfling E: The weak, fine-tuned binding of ubiquitous transcription factors to the IL-2 enhancer contributes to its T-cell-restricted activity. Nucleic Acids Res 1992, 20:2657-2665.

92. Hill CS, Treisman R: Transcriptional regulation by extracellular ee signals: mechanisms and specificity. Cell 1995, 80:199-211. This paper and [93] are outstanding reviews on the regulation of transcription factors by signal transcluction pathways, with emphasis on phosphorylation as a regulatory mechanism.

93. Hunter T, Karin M: The regulation of transcription by phospborylation. Cell 1992, 70:374-387.

94. Bram RJ, Hung DT, Martin PK, Schreiber SL, Crabtree GR: Identification of the immunophillns capable of mediating inhibition of signal transduction by cydosporin A and FKS06: roles of calcineurln binding and cellular location. Mol Cell Biol 1993, 13:4760-4769.

95. Milan D, Griffith ], Su M, Price ER, McKeon F: The latch region of calclneurin B is involved in both immunosuppressant-immunophilin complex docking and phosphatase activation. Cell 1994, 79:437-447.

342 Lymphocyte ac t iva t ion and ef fector funct ions

96. Liu J: FK506 and cyclosporin: molecular probes for studying intracellular signal transduction. Immunol Today 1993, 14:290-295.

97. Bierer BE, Hollander G, Fruman D, Burakoff SI: Cyclosporin A and FKS06: molecular mechanisms of immunosoppression and probes for transplantation biology. Curr Opin Immunol 1993, 5:763-773.

98. Flanagan WM, Corthesy B, Brain RJ, Crabtree GR: Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 1991, 352:803-807.

99. Frantz B, Nordby EC, Bren G, Steffan N, Paya CV, Kincaid RL, ** Tocci MJ, O'Keefe SJ, O'Neill EA: Calcineurin acts in synergy

with P/VIA to inactivate IKB/MAD3, an inhibitor of NF-KB. EMBO J 1994, 13:861-870.

This paper shows that pSO/p65 NFKB is also a target for calcineurin in activated T cells, and that the mechanism may involve phosphorylation and degradation of IKBo~.

100. McCaffrey PG, Kim PK, Valge-Archer VE, Sen R, Rao A: Cyclosporin A sensitivity of the NF-KB site of the IL2Rc( promoter in untransformed murine T-cells. Nucleic Acids Res 1994, 22:2134-2142.

101. Nghiem P, Ollick T, Gardner P, Schutman H: Interleukin-2 * transcriptional block by multifunctional Ca2÷/calmodulin

kinase. Nature 1994, 371:347-350. See annotation [102"].

102. Hama N, Paliogianni F, Fessler BI, Boumpas DT: Calcium/calm- , odulin-dependent protein kinase II downregulates both

calcineurin and protein kinase C-mediated pathways for c~okine gene transcription in human T-cells. J Exp Med 1995, 181:1217-1222.

Along with [101*] this paper suggests a role for calcium-mediated signalling pathways in downregulating IL-2 gene transcription.

103. Shahinian A, Pfeffer K, Lee KP, Kundig TM, Kishihara K, Wakeham A, Kawai K, Ohashi PS, Thompson CB, Mak TW: Differential T cell costimulatory requirements in CD28-deficient mice. Science 1993, 261:609-612.

104. Green JM, Noel PJ, Sperling AI, Walunas TL, Gray GS, Bluestone JA, Thompson CB: Absence of BT-dependent responses in CD28-deficient mice. Immunity 1994, 1:501-508.

105. Jenkins MK: The ups and downs of T cell costimulation. ** Immunity 1994, 1:443-445. See annotation [106**].

106. June CH, Bluestone IA, Nadler LM, Thompson CG: The B7 and ** CD28 receptor families. Immunol Today 1994, 15:321-331. This paper and [105**] review the importance of CD28/B7 and CTLA- 4/B7 interactions for initiating and shutting off the immune response.

107. Fraser JD, Straus D, Weiss A: Signal transduction events leading to T-cell lymphoklne gene expression. Immunol Today 1993, 14:357-362.

108. Umlauf SW, Beverly B, Kang S-M, Brorson K, Tran A-C, Schwartz RH: Molecular regulation of the IL-2 gene: rheoslalic control of the immune system. Immunol Rev 1993, 133:177-197.

109. Civil A, Geerts M, Aarden LA, Verweij CL: Evidence for a role of CD28RE as a response element for distinct mitogenic T cell activation signals. Eur J Immunol 1992, 22:3401-3403.

110. Williams TM, Moohen D, Burtein l, Roman, J, Bhaerman R, Godillot A, Mellon M, Rauscher FJ III, Kant JA: Identification of a zinc finger protein that inhibits IL-2 gene expression. Science 1991, 254:1791-1794.

111. Hoey T, Ya-Lin S, williamson K, Xu X: Isolation of two new members of the NF-AT gene family and functional characterization of the NF-AT proteins. Immunity 1995, 2:461-472.

112. Serfling E, Avots A, Neumann M: The architecture of the interleukin-2 promoter: a reflection of T lymphocyte activation. Biochim Biophys Act, 1995, 1263:in press.

j lain, c Loh and A Rao, Division of Cellular and Molecular Biology, B446, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.

E-marl: Anj [email protected]

E-marl: [email protected]

E-mail: [email protected]

transcriptional regulation of the il-2 gene

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