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On the stochastic regulation of interleukin-2 transcription
seminars in I M M U N OL OG Y, Vol 11, 1999: pp. 357]367 Article No. smim.1999.0192, available online at http:rrwww.idealibrary.com on
On the stochastic regulation of interleukin-2 transcription .. Georg A. Hollander
Interleukin-2 (IL-2) is a growth and differentiation factor critical for clonal T cell expansion and function. Produced exclusively in T cells, IL-2 transcription and synthesis occurs only after appropriate cellular activation via the clonotypic antigen]receptor and co-stimulatory molecules. IL-2 gene expression is initiated by the cooperative binding of different transcription factors and is predominantly controlled at the transcriptional level. Recently, it has been demonstrated that IL-2 transcriptional activity is normally confined to a single, randomly chosen allele. This monoallelic expression of a non-receptor gene product encoded at a non-imprinted, autosomal locus represents an unusual regulatory mode. Although the molecular mechanisms operational for IL-2 transcription have yet to be defined, allele-specific expression of the IL-2 locus constitutes an important expansion to the concept of stochastic gene expression.
belongs to a family of short-chain four helical bundle cytokines2 which share the common cytokine receptor g chain Žg c. for signal transduction.3 The molecular analysis of IL-2 regulation has become a paradigm of how cytokine expression is controlled. Although not produced by resting T cells, antigen stimulation of mature T cells results in the de novo synthesis of IL-2. The interaction of IL-2 with its cognate high-affinity receptor can occur either in an autocrine or a paracrine fashion and thus regulates both the magnitude and duration of an IL-2 restricted immune response. As a result, transcription and synthesis of IL-2 are often used as key indicators for the successful activation of T cells. Depending on the nature of the responding cell, IL-2 signalling results in a broad range of different biological effects. For T cells, IL-2 promotes the progression from the G1 stage to the S phase of the cell cycle and thus serves as an essential factor for the clonal expansion of newly activated cells.4 Immature thymocytes have also been demonstrated to express IL-2 during distinct stages of their intrathymic development. For example, IL-2-specific mRNA has been detected in murine thymoyctes as early as day 13 of embryogenesis5 and is also readily apparent in thymic tissue of neonatal mice.6 IL-2 exerts also an important effect on the differentiation of cells other than T cells. Signalling via IL-2 increases the immunoglobulin synthesis and J-chain transcription of B lymphocytes;7 potently activates the cytolytic activity of natural killer ŽNK. cells;8 induces lymphokine-activated killer cells;8 promotes proliferation, differentiation and cytolytic capacity of macrophages;9,10 enhances the antibodydependent tumoricidal activity of monocytes; and functions as a trophic factor for stroma cells of the thymic microenvironment.11 Moreover, IL-2 is critically important for the immune system in general as mice homozygous for a targeted disruption of the IL-2 locus display multiple irregularities in their response to self-antigens12 and to a range of different viral antigens.13,14 Similarly, impaired IL-2 secretion
Key words: interleukin-2 r transcription factors r T cells r monoallelic expression Q1999 Academic Press
Introduction EXTENSIVE COMMUNICATIONS between lymphocytes, antigen-presenting cells and stromal cells are required for a normal immune system to generate an antigen-specific response. This interaction is mediated concurrently by direct cell]cell contacts via a panoply of cell surface molecules and an array of soluble mediators, collectively called cytokines. Interleukin-2 ŽIL-2. is a prominent cytokine and acts as a major growth factor for the clonal expansion of T cells.1 Together with IL-4, IL-7, IL-9 and IL-15, IL-2
From the Pediatric Immunology Department of Research and The Children’s University Hospital, Hebelstrasse 20, 4031 Basel, Switzerland. E-mail: [email protected] Q1999 Academic Press 1044-5323r 99 r 050357q 11 $30.00r 0
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by activated T cells has been associated with functional immunological defects in man.15 ] 18 The importance of IL-2 in maintaining normal T cell homeostasis has been highlighted by the observation that IL-2 is a critical determinant involved in programmed cell death.19 In vivo, antigen receptor stimulation of mature ab TCRq lymphocytes previously exposed to IL-2 leads to apoptosis while the concomitant blockade of IL-2 with antibodies reverses this marked deletion. Thus, IL-2 may also participate in a feedback regulatory mechanism by predisposing mature T lymphocytes to apoptosis. In contrast, IL-2 overproduction results in the loss of T cell anergy in vitro 20 and causes the functional enhancement of self-reactive T cells in vivo leading to autoimmunity.21 Based on these numerous observations the conclusion may be drawn that IL-2 synthesis has to be tightly regulated in order to assure normal immune homeostasis. This contention is further reflected by the fact that IL-2 gene expression is regulated almost exclusively at the transcriptional level where three different but partially interrelated mechanisms are operational: Ži. the dynamic and cooperative assembly of diverse transcription factors; Žii. cell-type specific alterations in chromatin structure; and Žiii. a mechanism of monoallelic transcription. Combined, these diverse mechanisms guarantee that T cell lineage- and activation-specific IL-2 expression will occur in response to a variety of biochemical signals but in accordance with a discriminatory threshold.
torŽs. via a signaling pathway which is functional only in T cells. A third possibility assumes that activation dependency and cell-type specificity are mediated by two separate mechanisms: Transcriptional activation is induced in response to physiological stimuli Žwhich do not have to be T cell specific. but T cells have specifically altered their chromatin to accommodate for IL-2 gene accessibility. IL-2 regulation was first confined to a minimal IL-2 DNA regulatory sequence mapped to the first 300 base pairs Žbp. proximal to the start site.23 Further analysis characterized the organization of this transcriptional control element and defined extensive sequence homologies between mouse and man.24 Within this minimal IL-2 promoterrenhancer, several binding sites have been defined for tissue restricted and ubiquitously expressed transcription factors including NF-ATs, AP-1, NF-k B, and Oct-1, respectively Žreviewed in refs 22,25,26.. Some of these factors interact with their target DNA sequence without prior activation Že.g. Oct-1. while the majority of transcriptional activators implicated in IL-2 transcription associate with their DNA binding sites only after specific activation Že.g. NF-AT, AP-1, NF-k B..27 ] 29 Although these transcription factors allow for the induction of an IL-2 reporter gene in T-lymphocytes, none of these factors are lineage-specific. Moreover, the individual regulatory sequences within the IL-2 promoter are non-consensus sites that bind their cognate factors with an up to 10-fold lower affinity than the corresponding consensus sites.30 Importantly, mutations of each of these sites to the corresponding consensus sequences results in the loss of T cell transcriptional specificity demonstrating that the binding affinity of ubiquitous factors contributes Žat least in part. to the cell-type specific activity.30 To date, none of the factors necessary for IL-2 expression using the minimal enhancerrpromoter sequence has been demonstrated to be T cell-specific. However, this conclusion does not exclude the possibility that T cell lineage-specific protein]DNA interactions outside of the minimal promoterrenhancer account for ‘architectural’ chromatin changes which ‘sensitize’ the IL-2 locus to the binding of activationinduced transcription factors. The contention that long range interactions may be operational well beyond the positively regulating proximal DNA sequences has been put forth by the above results and by analysis of transgenic mice. The use of a construct containing the E. coli lac Z reporter gene under the control of a 583 bp promoterrenhancer sequence immediately upstream of
Regulatory sequence elements within the IL-2 gene The restriction of IL-2 expression to activated T cells provides evidence for a regulatory mechanism driven by two kinds of information: Ži. transient changes in the provision of biochemical signals activate factors which control IL-2 transcription; and Žii. lineagespecific determinants impose a tissue restriction for the control of IL-2 expression. Several possibilities have been suggested that bring these two mechanisms together and that allow for the IL-2 gene transcription to occur.22 One scenario predicts that IL-2 expression depends on at least one constituatively expressed transcription factor that is T cellspecific but has to interact with activation-induced factors in order to initiate IL-2 expression. A second model foresees that T cell-restricted IL-2 production is exclusively dependent on the activation of Ža. fac358
On the stochastic regulation of interleukin-2 transcription
the IL-2 start site was not sufficient to convey in vivo a position independent expression since only one of 17 transgenic mice expressed this reporter gene.31 Molecular investigations have thus not yet identified a moderately sized genomic sequence sufficient to regulate IL-2 gene expression in an inducible but lineage-restricted and integration independent manner. Despite this shortcoming, extended analysis of 2500 bp 59 of the minimal promoterrenhancer identified sequences which either intensify or suppress the magnitude of an inducible murine IL-2 response.24 Deletions within this distal cis-element yet reveal that no region of this sequence altered either the requirement for induction, the kinetics of stimulation, nor the cell-type specificity of IL-2 expression. Even in the presence of extensive 59 flanking sequences, reporter gene transcription remains strongly dependent on the factors binding within the first 300 bp upstream of the start site stressing the conclusion that the additional 59 sequences exert at most a secondary modulating effect on IL-2 transcription. The observation that optimal cytokine production, especially in naıve ¨ T cells, requires signaling via the TCR and engagement of co-receptors raises the possibility that only activated T cells generate a specific transcription factor which accounts for cell- and activation-restricted IL-2 transcription. ŽAlternatively, costimulatory signals may induce transcription by altering the molecular composition of transcription factors as has been shown for AP-1.32 . Engagement of the surface protein CD28 provides co-stimulatory signals critical for the integral activation of T cells and the subsequent production of IL-2. CD28-mediated signals exert their stimulatory effect via a sequence designated the CD28 response element ŽCD28RE..33,34 CD28RE constitutes a variant NF-k B site 35 and is adjacent to and functionally cooperates with an AP-1 binding sequence.36 These findings preclude the existence of a T cell-specific transcription factor that exclusively integrates CD28 Žor other co-receptor. mediated signals. Although tissue-specific repressors of IL-2 transcription have not yet been identified, several lines of evidence suggest that different DNA binding factors may act as negative regulators Že.g. p50 of the family of NF-k B molecules, ZEB, Ets-1 and others.. A mechanism of silencing could contribute in several ways to the documented restriction in IL-2 transcription: Ži. tissue specificity; Žii. silencing of IL-2 expression in fully differentiated Th2 cells; and Žiii. loss of IL-2 transcription in anergic T cells. Mutational analyses of the IL-2 promoterrenhancer have defined a re-
pressor element that overlaps with a negative regulatory element, designated NRE-A.37,38 Binding of the highly conserved ‘two-handed’ zinc finger protein ZEB to NRE-A has been implicated in the silencing of the IL-2 gene in activated Th2 cells.39 However, this repressor factor is not T cell-specific.40 Moreover, T cells rendered anergic display an altered composition of their transcription factors, which most likely effect IL-2 repression in anergic T cells. For example, in vivo anergized CD4q T cells have been demonstrated to display severely perturbed levels of AP-1 and FosrJun-containing NF-AT complexes and NF-k B complexes composed of the transcriptionally inactive p50 homodimer.41,42 The available data concerning the role of transcription factors governing IL-2 expression suggest that the cooperative binding of individual factors to weak sites within the promoterrenhancer renders the entire complex competent for lineage-specific and activation induced IL-2 transcription. A requirement for stereospecific contacts among distinct transcription factors themselves may be of additional importance. Moreover, in vivo footprinting has implied that nuclear factors fail to interact with the minimal promoterrenhancer in unstimulated cells and non-T cells,43 suggesting additional epigenetic features specific in activated T cells. Thus, gene transcription is not the mere sum of multiple independent transcription factors binding to their corresponding DNA sequence. Rather, transcriptional activation is determined by an architectural assembly of interactive nucleoproteins which form distinct protein]DNA and protein]protein complexes that execute a precise IL-2 regulation at the minimal promoterr enhancer.44
Regulation of IL-2 transcription by chromatin changes Histones, linker histones, high molecular group ŽHMG. molecules and other proteins package the eukaryotic DNA into distinct nucleosomal arrays. This organization of the genome into nucleoprotein complexes is essential for the expression and silencing of genetic material, and thus acts as a major regulator in development. Moreover, the exact position of a gene with regards to DNA packaging proteins influences the expression level as DNA binding proteins impart a different stability to the wrapping of DNA.45 Hence, chromatin architecture plays a key role in the events leading to transcription46 and alterations in the chro359
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matin structure have thus been proposed to govern the T cell-restricted22 and possibly allele-specific IL-2 expression. A direct approach to determine the organization of the nucleoprotein architecture is to use in vivo DNase I digestion of chromosomal DNA. This nuclease analysis can identify distortions in the tertiary structure of the DNA helix induced by protein binding. It is of note that DNase I hypersensitive sites are not necessarily histone-free but may actually inflect the recruitment of nuclease by transcription factors bound to an intact nucleosomal infrastructure.47 In vivo DNase I footprinting of sequences y600 to q400 relative to the IL-2 start site reveals the existence of several, tissue-specific and stimulation independent differences in the DNA structure.48 These hypersensitive sites ŽHS. are confined to a 59 sequence of the fragment analyzed. Importantly, these distinct HS of resting T cells do not repress IL-2 gene transcription as they are equally present in activated T cells. Tissue-specific DNA]protein interactions have also been directly established for a sequence centered around residue y410.48 This particular site is recognized by HMG proteins which, in turn, may cooperate with other factors present in the nucleus to form specific complexes. These proteins may alter the overall architecture of the 59 sequence with regard to the minimal promoterrenhancer located at nucleotides y300 to y45 relative to the IL-2 start site. The interaction of HMG or other nucleoproteins acting as ‘lineage’ specific, constitutive marking proteins with DNA sequences beyond residue y390 could ‘open’ the chromatin of the minimal promoterrenhancer and render this regulatory sequence sensitive to activation by transcription factors. Upon successful T cell stimulation, additional DNase I HS can be noticed within the region of the IL-2 promoterrenhancer revealing the induction of activation-induced changes in chromatin conformation.48 These latter alterations are Žmost likely. caused by the binding of transcription factors to their respective binding sites. Studies focusing on chromatin structure have not addressed yet the issue of position independence of the IL-2 regulatory sequence. Although in vivo footprinting has identified tissue specific chromatin features within 600 bp 59 to the start site, the use of an almost identical sequence as a transgene has not conveyed protection against position effects.31 Thus, the existence of genomic sequences further 59 may mark the boundary of the active chromatin domain and may function as an insulator. In this context, it
has recently been shown that mammalian insulator sequences can prevent the loss of transcription of a reporter gene, which usually adopts the epigenetic hallmarks of an inactive gene,49,50 i.e. nuclease inaccessibility, DNA hypermethylation and histone hypomethylation. Taken together, these descriptive studies of chromatin structure in vivo have so far delineated that the IL-2 gene adopts a T cell-lineage specific architecture which is further altered upon lymphocyte activation. These findings fulfill the general demand that celltype specific and induction-dependent chromatin changes regulate IL-2 transcription. Accordingly, chromatin remodeling of IL-4 and IL-13 genes has recently been demonstrated and these changes correlate with a differentiation to a Th2 effector phenotype.51
Monoallelic expression of IL-2 Monoallelic expression is an epigenetic phenomenon in which only a single copy, or allele, of a given gene is expressed. This is unusual in that most genes of diploid organisms are expressed equally from each copy Žwith X chromosome encoded genes constituting a notable exception.. Monoallelic gene transcription has recently been demonstrated as a third regulatory mechanism operational in the control of IL-2 transcription.52 Although immature thymocytes have an ‘open’ IL-2 locus and have been shown to transcribe IL-2,5 it is presently not known whether monoallelic expression is already fixed at this developmental stage or whether this mode of transcriptional regulation is established later. Initially it was observed that the bulk of peripheral T cells heterozygous for a null mutation of the IL-2 gene ŽIL-2qry . produced only half as much IL-2 when compared to wild type ŽIL-2qrq . T cell cultures. This result could be accounted for by two mutually exclusive models: First, each of the T cells in both cultures produce IL-2 but, when analyzed at a single cell level, a IL-2qry T cell synthesizes only half as much of the cytokine in contrast to a IL-2qrq T cell. Alternatively, the pool of IL-2qry T cells consists of two equally sized subgroups, that is one population that produces IL-2 and the other group that fails to do so. This latter model posits two testable properties: first, the frequency of IL-2 secreting cells among IL-2qry T lymphocytes has to be half of that noticed for wild type T cells, and second, IL-2 producing T cells bearing only one functional IL-2 allele produce iden360
On the stochastic regulation of interleukin-2 transcription
tical quantities of the cytokine per single cell when compared to T cells with two functional alleles. The analysis of mature thymocytes and peripheral T cells heterozygous for an IL-2 null mutation revealed in young mice that only approximately half of all the cells produced IL-2 upon stimulation.52 Moreover, single cell quantification of intracytolplasmic IL-2 by use of digital analysis with an interactive laser cell cytometer demonstrated that heterozygous but IL-2 synthesizing T cells did produce the cytokine as copiously as wild type T cells. The similar frequency of IL-2 producing versus IL-2 non-producing mature thymocytes and peripheral T cells in young heterozygous mice argues for a random process of silencing of one of the IL-2 loci and concurrently argues against a control feed-back mechanism which assures a preferential or even exclusive transcription from the functional allele. Moreover, these results also demonstrate that the autocrine use of IL-2 is not a decisive element for intrathymic T cell maturation. In contrast, analysis of older mice demonstrated that IL-2qry peripheral T cells competent to secrete IL-2 have a growth advantage over heterozygous cells which fail to express this cytokine secondary to silencing of their functional allele.52 This result implies that the silencing of one of the two IL-2 alleles is stable for each clonal progeny and thus does not appear to be ‘negotiated’ anew with every cell division. ŽHowever, formal proof of this assertion is presently not available because ‘allelic fidelity’ of IL-2 silencing has not yet been reported at the clonal level.. Considering the situation of wild type ŽIL-2qrq . T cells, the con-
clusion that IL-2 is regulated in an allele-specific manner is further underscored by single cell RT-PCR analysis. Single T cells with allele-specific IL-2 sequence differences express usually either the maternal or the paternal allele but not both. Taken together, these cellular and molecular analyses in primary murine T cells argue for a monoallelic control of IL-2 transcription. The monoallelic transcription of the IL-2 locus was recently questioned in an experiment using transgenic mice where the first exon of the IL-2 gene was replaced with cDNA encoding the green fluorescent protein ŽGFP..53 Upon activation of T lymphocytes heterozygous for this ‘knock-in’, concomitant expression of IL-2 and GFP is readily visible in approximately 60% of single cells analyzed. Among the remaining T cells a majority expresses exclusively IL-2 while a small fraction of cells is positive only for GFP. This finding is difficult to reconcile with monoallelic expression of IL-2 as long as the molecular mechanisms regulating transcription from only one of the two alleles is not determined. However, the loss of exon 1 of the IL-2 locus, the introduction of GFP cDNA and the insertion of polyadenylation sequences may account for the disturbance of regulatory elements by either removal or the inclusion of regulatory sequences.
Monoallelic expression of other cytokines Two independent experimental approaches recently
Table 1. Characteristics of monoallelic gene expression Žn.d.s not determined. Gene
Replicative asynchrony
Time of establishment
X-linked genes in females
Yes
Early in embryogenesis
Imprinted genes
Yes
T and B cell receptor genes
Parental origin of silent allele
Allelic difference in chromatin structure or factor binding
Requirement for functional gene product Žfeed back.
Random
Yes
No
Gametogenesis
Non-random
Yes
No
n.d.
Lymphopoiesis
Random
Yes
Yes
NK cell receptor genes
n.d.
n.d.
Random
n.d.
n.d.
Olfactory genes
Yes
Early in embryogenesis
Random
n.d.
n.d.
IL-2 gene
Yes
n.d.
Random
n.d.
No
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documented that IL-4 transcription is also controlled in an allele-specific manner. Analysing activated wild type T cells biased for a Th2 phenotype and bearing two IL-4 alleles which allow assignment of the parental origin revealed that each of the two alleles is regulated independently with transcription occurring preferentially from a single allele.54 The second model took advantage of a mutant mouse in which one of the IL-4 alleles was replaced by the human CD2 cell surface molecule.55 Upon stimulation, most of the cells from these transgenic mice expressed only IL-4 or the CD2 reporter gene, thus revealing the stochastic nature of IL-4 transcription. Interestingly, the increased probability of allelic transcription Žand thus the loss of mono-allelic expression. was directly correlated to the strength of the activation signal delivered via the TcR. In addition to IL-4, also the transcription of GM-CSF appears to be regulated via a stochastic process.54 Thus, allele-specific activation may reflect a general feature of cytokine transcriptional regulation.
Interestingly, X inactivation is mediated by a posttranscriptional mechanism, i.e. the stabilization of Xist RNA, rather than by the regulation of the Xist promoter. Imprinting reflects the silencing of an allele in a heritable and parental-specific manner.64 Established already during gametogenesis, imprinted genes are frequently clustered within the genome and are associated with asynchronous replication during S-phase of the cell cycle Žreviewed in ref 65.. From the onset, imprinting has been associated with differential DNA methylation for most but not all of the genes.66 However, it remains to be determined whether the role of methylation is to act as the gametic imprinting signal or whether it merely maintains allele-specific expression. Interestingly, in some instances, imprinting is restricted to only certain organs suggesting the existence of tissue-specific transacting factorŽs..67 Experimental evidence proposes several additional mechanisms to be operational for imprinting to occur including the existence of chromatin insulators; transcriptional and post-transcriptional interference with anti-sense RNA; and enhancer competition. In each of these models, allele-specific DNA methylation is either directly or indirectly responsible for gene silencing. The genes for the antigen receptors of B and T lymphocytes undergo recombinations during distinct stages of maturation. The productive chromosomal rearrangement of one allele yields a functional receptor chain which, using a feed-back mechanism, precludes the second allele to undergo chromosomal rearrangement.68 Trans-acting proteins, enhancers and cis-regulatory sequences mediate local changes in chromatin structure rendering the loci of the differ ent antigen receptor chains accessible to the recombination apparatus. In vitro experiments have demonstrated that DNA methylation inhibits69 and that undermethylation is required for the rearrangement of B and T cell antigen receptor genes.70 These findings are compatible with the hypothesis that demethylation directs the initial choice of a single allele for rearrangement. The family of murine Ly49 NK cell receptors are MHC class I specific antigen receptors belonging to the C-type lectin superfamily. Monoallelic expression of these receptors generates a diverse repertoire of NK cells with restricted specificities as single NK cell receptors recognize different MHC class I molecules.71 Expression of these receptors in mice heterozygous for the Ly49A, Ly49C and Ly49G2 gene is usually effected from one or the other allele and
Comparative characteristics of monoallelic gene expression In addition to the IL-2 and IL-4 genes, other examples of allele-specific expression include X-inactivation,56 parental imprinting,57 the monoallelic exclusion of the antigen receptors of T and B lymphocytes and the stochastic selection of both natural killer ŽNK. 58,59 and olfactory receptors.60 Although bearing some common features, the mechanisms responsible for these forms of monoallelic gene expression appear to be distinct from the characteristics of the IL-2 locus Žsee Table 1.. X-inactivation assures the correct gene dosage for cells of different gender so that female cells will express the same number of X-linked genes as male cells. This phenomenon of gene dosage compensation results from mechanisms that first count the chromosome number and then inactivate the supernumerous X-chromosomeŽs..60,61 The counting mechanism allows for only one of the X-chromosomes to be active and this task may be achieved randomly by a trans-activating ‘blocking’-factorŽs.. The Xist gene, which maps to the X chromosome inactivation center ŽXic. and encodes an untranslated RNA, is required for initiation, propagation and maintenance of the inactivation process.62 A process of DNA methylation is responsible for the silencing of the Xist gene on the inactive X-chromosome.63 362
On the stochastic regulation of interleukin-2 transcription
only rarely from both loci.58,59 Monoallelic Ly49 gene expression appears to arise as a consequence of a stochastic Ly49 gene activation mechanism although the molecular details responsible for it have not yet been elucidated. The olfactory receptor gene family contains approximately 1000 genes, which are clustered within multiple loci that are broadly distributed in the genome. These loci lie within paralogous chromosomal regions that appear to have arisen by duplications of large chromosomal domains followed by extensive gene duplication and divergence.72 In individual olfactory neurons, the expression of a given receptor derives exclusively from one allele and only one allelic array encoding multiple receptor genes is active.73 Alleles encoding the odorant receptors are replicated asynchronously, further emphasizing the phenomenon of allelic inactivation. This random inactivation of one allelic array and the concomitant cis-control of the active allele are established early in development and may also affect cells of non-neuronal lineage.73 A cis stochastic model of olfactory gene regulation has been proposed to control first allelic inactivation which is then followed by mechanisms that chose first one of the several chromosomal arrays on a single allele and then a single receptor.73 The precise molecular mechanisms operational in this allele-specific expression are, however, not known to date. Expression of the IL-2 gene is}as already alluded to}in many aspects different when compared to the examples outlined above: Ži. IL-2 is present as a singular copy located on mouse chromosome 3 Žposition 19.2.;74 Žii. this locus is not known to be imprinted;75 Žiii. IL-2 expression is independent of recombination;76 and Živ. there is no feed-back mechanism operational which ensures that the expression from a defective allele encoding IL-2 is switched to the functional allele providing a normal gene product.52
lated by at least two conceptually different mechanisms. One model could predict that a single IL-2 allele is randomly marked Žfor example by differential methylation. and consequently silenced by a molecular mechanism either similar or identical to those modeled for the phenomenon of imprinting. Alternatively, transcription of IL-2 may be achieved by a limited concentration of specific transcription factors, which allows at any given time for the occupancy of only one of the two promoters of the IL-2 locus. The inverse correlation between DNA methylation and tissue-specific gene expression has been established for in vivo transcription.77 The covalent modification of cytosine to methyl-cytosine may change the chromatin structure and consequently the binding of the transcriptional machinery to the double helix.78,79 Such a structural modification controls transcriptional gene activity either at a local Ži.e. monogenetic. level or, alternatively, may influence many genes within a chromosomal stretch of variable length. The correlation between demethylation and activation of genetic loci has been observed for several genes in the immune system,80 including the IL-4 locus. This latter example is relevant to the discussion here because the IL-4 locus is hypermethylated in naıve ¨ T cells but not in polarized Th2 cells81 where IL-4 expression occurs frequently in a monoallelic manner.54,55 Following initial stimulation and demethylation, the ensuing chromatin remodeling of the IL-4 locus proceeds Žor at least parallels. transcriptional activity.82,51 This change to an ‘open’ chromatin configuration Žnow responsive to transcriptional activation. may very well correspond to the structural alterations seen in the IL-2 locus of activated T cells.48 Whether demethylation andror chromatin changes occur selectively for only one of the two alleles of activated primary T cells has not yet been directly evaluated. Circumstantial evidence suggests, however, that there is a difference in chromatin structure between the active and the silent IL-2 allele as asynchronous DNA replication has been reported for this locus in dividing primary T cells.52 Asynchrony of replication has previously been shown for imprinted and X chromosome inactivated genes but not for most other genes with the notable exception of the olfactory gene.83 ] 86 Although an allelespecific difference in methylation has not yet been reported for the IL-2 locus, differential methylation has been documented where the silenced allele is methylated while the expressed allele of an imprinted gene is not.67,87 Marking by differential methylation
Models for monoallelic IL-2 expression The precise molecular mechanisms regulating monoallelic IL-2 expression are presently unknown. However, any model explaining monoallelic IL-2 transcription not only needs to clarify the stochastic nature of this process but has to accommodate the particular characteristics of the IL-2 locus and its transcriptional activation restricted to T cell stimulation. Basically, monoallelic expression may be regu363
G. A. Hollander ¨
andror the presence of a specific DNA binding protein affecting only one of the two alleles may be the discriminatory process operational in the monoallelic control of the IL-2 locus. A second model explaining monoallelic transcription of the IL-2 locus could foresee that allele-specific transcription is regulated probabilistically by the correct combination of transcription factors. Although similar to the mode of regulation of most genes, this model stipulates that the stochastic activation of only one of the two alleles is entirely dependent on a limited concentration of the transactivating complex. Thus, at any given time during IL-2 transcription and possibly largely independent of the degree of a stimulus, the factors available will only be sufficient to transactivate one of the two promoters. Disregarding physical constraints related to the positioning of the alleles within the nuclear architectural organization, an extreme prediction of this model would then anticipate that a single transcription complex is sufficient to initiate transcription in an activated T cell. In general terms, this second model also provides the mechanistic basis for unstable gene expression, a genetic pattern where a particular locus is transcribed only intermittently and preferentially from a single allele Žreviewed in ref 88]90.. Observations in cloned T cells, where the initial activation of an IL-2 reporter construct represents a probabilistic event, provide experimental support for such a second model.91 There are, however, several findings regarding IL-2 transcription, which are difficult to reconcile with this second model. Foremost, analysis of single F1 T cells bearing two IL-2 alleles separable by parentalspecific sequences have not revealed within 10 h after stimulation a bi-allelic expression at the RNA level. Normally, IL-2 transcripts can be detected as early as 2 h after mitogenic activation of primary resting T cells. They are at their highest levels between 4 and 8 h after stimulation and decline subsequently to be completely absent by 24 h.53,92 These results imply that if unstable IL-2 gene expression were to occur, the transcriptional machinery would have to occupy a single allele for an extended period of time Ži.e. ) 8 h.. There is also an alternative explanation for this result: if the half-life of the IL-2 specific message were extremely small}that is considerably shorter than the reaction kinetics of transcription itself}a comparable outcome would be anticipated. The observed rate of increase of IL-2 transcripts in activated T cells invalidates, however, this explanation92 if one does not postulate at the same time that the transcriptio-
nal frequency of a single allele is considerably higher within the first 4]8 h after stimulation when compared to earlier or later time points. An other result is also not apparently compatible with a model of slow-unstable gene expression from a single functional allele.90 The digital quantitation of intracytoplasmic IL-2 in activated T cells from IL-2qrq and IL-2qry mice reveals the same range of cytokine concentrations per single cell. Finally, T cells from old mice heterozygous for a null mutation reveal an obvious increase in the frequency of IL-2 expressing cells 52 which suggests a fixed allelic pattern of transcription Žas described for IL-4 producing clones54 .. Therefore, any mechanism regulating monoallelic transcription of cytokines will need to explain the symmetric segregation to the sister chromatids during cell division. A model of limited availability of transcription factors results in a random choice of a single allele but fails to easily explain an allelic pattern of IL-2 expression that is transmitted as a stable epigenetic trait
Conclusion The purpose of cytokines being expressed in a monoallelic manner remains at present ill-explained but is certainly of teleologic interest. For the IL-4 locus it was argued that a probabilistic expression of only one allele allows a combinatorial assortment of distinct cytokine genes among the clonal progeny of T cells with an identical antigen receptor specificity.54 There is still another evolutionary explanation to be considered. Avoiding transcription from both IL-2 alleles, the organism takes advantage of diploidy. Such a restriction to monoallelic usage may bear the benefit that IL-2 isoforms can be functionally sampled during an immune response as different IL-2 sequences have been described encoding variable biological activities.93 Consequently, single T cells expressing a biologically superior isoform will have a growth advantage which ultimately benefits the entire individual. A monoallelic mechanism will also diminish the representation of serious recessive mutations at the clonal level and may thus be decisive for the outcome of an immune response. However, critical for any attempt of an explanation is the issue whether a twofold difference in the in vivo expression of a cytokine has a functional impact on a system as complex as the immune system. Results obtained in an allogenic transplantation model support the in vivo importance of such a relatively small variance in 364
On the stochastic regulation of interleukin-2 transcription
12. Sadlack B, Merz H, Schorle H, Schimpl A, Horak I Ž1993. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75:253]261 13. Cousens LP, Orange JS, Biron CA Ž1995. Endogenous IL-2 contributes to T cell expansion and IFN-gamma production during lymphocytic choriomeningitis virus infection. J Immunol 155:5690]5699 14. Kundig TM, Schorle H, Bachmann MF, Hengartner H, Zinkernagel RM, Horak I Ž1993. Immune responses in interleukin-2-deficient mice. Science 262:1059]1061 15. Chatila T, Castigli E, Pahwa R, Pahwa S, Chirmule N, Oyaizu N, Good RA, Geha RS Ž1990. Primary combined immunodeficiency resulting from defective transcription of multiple T-cell lymphokine genes. Proc Natl Acad Sci USA 87: 10033]10037 16. Doi S, Saiki O, Tanaka T, Ha-Kawa K, Igarashi T, Fujita T, Taniguchi T, Kishimoto S Ž1988. Cellular and genetic analyses of IL-2 production and IL-2 receptor expression in a patient with familial T-cell-dominant immunodeficiency. Clin Immunol Immunopathol 46:24]36 17. Pahwa R, Chatila T, Pahwa S, Paradise C, Day NK, Geha R, Schwartz SA, Slade H, Oyaizu N, Good RA Ž1989. Recombinant interleukin 2 therapy in severe combined immunodeficiency disease. Proc Natl Acad Sci USA 86:5069]5073 18. Weinberg K, Parkman R Ž1990. Severe combined immunodeficiency due to a specific defect in the production of interleukin-2. N Engl J Med 322:1718]1723 19. Lenardo MJ Ž1991. Interleukin-2 programs mouse alpha beta T lymphocytes for apoptosis. Nature 353:858]861 20. Essery G, Feldmann M, Lamb JR Ž1988. Interleukin-2 can prevent and reverese antigen-induced unresponsiveness in cloned human T lymphocytes. Immunology 64:413]417 21. Andreu-Sanchez JL, Alboran I, Marcos MAR, Sanchez-Movilla A, Martinez-A C, Kroemer G Ž1991. Interleukin-2 abrogates the nonresponsive state of T cells expressing a forbidden T cell receptor repertoire and induces autoimmune disease in neonatally thymectomized mice. J Exp Med 173:1323]1329 22. Rothenberg EV, Ward SB Ž1996. A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin 2 gene regulation. Proc Natl Acad Sci USA 93:9358]9365 23. Fujita T, Shibuya H, Ohashi T, Yamanishi K, Taniguchi T Ž1986. Regulation of human interleukin-2 gene: functional DNA sequences in the 59 flanking region for the gene expression in activated T lymphocytes. Cell 46:401]405 24. Novak TJ, White PM, Rothenberg EV Ž1990. Regulatory anatomy of the murine interleukin-2 gene. Nucleic Acids Res 18:4523]4533 25. Jain J, Loh C, Rao A Ž1995. Transcriptional regulation of the IL-2 gene. Curr Opin Immunol 7:333]342 26. Serfling E, Avots A, Neumann M Ž1995. The architecture of the interleukin-2 promoter: a reflection of T lymphocyte activation. Biochim Biophys Acta 1263:181]200 27. Serfling E, Barthelmas R, Pfeuffer I, Schenk B, Zarius S, Swoboda R, Mercurio F, Karin M Ž1989. Ubiquitous and lymphocyte-specific factors are involved in the induction of the mouse interleukin 2 gene in T lymphocytes. Embo J 8:465]473 28. Brunvand MW, Schmidt A, Siebenlist U Ž1988. Nuclear factors interacting with the mitogen-responsive regulatory region of the interleukin-2 gene. J Biol Chem 263:18904]18910 29. Rooney JW, Sun YL, Glimcher LH, Hoey T Ž1995. Novel NFAT sites that mediate activation of the interleukin-2 promoter in response to T-cell receptor stimulation. Mol Cell Biol 15:6299]6310 30. Hentsch B, Mouzaki A, Pfeuffer I, Rungger D, Serfling E Ž1992. The weak, fine-tuned binding of ubiquitous transcription factors to the Il-2 enhancer contributes to its T cell-restricted activity. Nucleic Acids Res 20:2657]2665
IL-2 production. Analysing Graft-versus-Host disease ŽGVHD. in lethally irradiated mice reconstituted with both allogenic bone marrow cells and T cells bearing two or one functional IL-2 allele, survival could be directly correlated to the IL-2 gene dosage ŽG. Hollander, manuscript in preparation.. These results ¨ demonstrate that a two-fold difference in IL-2 expression has a definite bearing on the outcome of an immune response. This is not entirely unexpected as relatively small concentration gradients of growth and differentiation factors constitute a central principle in developmental biology. The elucidation of the molecular mechanisms governing allele-specific IL-2 transcription becomes therefore the more important.
Acknowledgements I would like to thank E. Palmer and N. Iscove for helpful discussions and review of the manuscript. This work was supported by grants from the Swiss National Science Foundation Ž31’43600.95 and 3239-055233.98r1..
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On the stochastic regulation of interleukin-2 transcription .. Georg A. Hollander
Interleukin-2 (IL-2) is a growth and differentiation factor critical for clonal T cell expansion and function. Produced exclusively in T cells, IL-2 transcription and synthesis occurs only after appropriate cellular activation via the clonotypic antigen]receptor and co-stimulatory molecules. IL-2 gene expression is initiated by the cooperative binding of different transcription factors and is predominantly controlled at the transcriptional level. Recently, it has been demonstrated that IL-2 transcriptional activity is normally confined to a single, randomly chosen allele. This monoallelic expression of a non-receptor gene product encoded at a non-imprinted, autosomal locus represents an unusual regulatory mode. Although the molecular mechanisms operational for IL-2 transcription have yet to be defined, allele-specific expression of the IL-2 locus constitutes an important expansion to the concept of stochastic gene expression.
belongs to a family of short-chain four helical bundle cytokines2 which share the common cytokine receptor g chain Žg c. for signal transduction.3 The molecular analysis of IL-2 regulation has become a paradigm of how cytokine expression is controlled. Although not produced by resting T cells, antigen stimulation of mature T cells results in the de novo synthesis of IL-2. The interaction of IL-2 with its cognate high-affinity receptor can occur either in an autocrine or a paracrine fashion and thus regulates both the magnitude and duration of an IL-2 restricted immune response. As a result, transcription and synthesis of IL-2 are often used as key indicators for the successful activation of T cells. Depending on the nature of the responding cell, IL-2 signalling results in a broad range of different biological effects. For T cells, IL-2 promotes the progression from the G1 stage to the S phase of the cell cycle and thus serves as an essential factor for the clonal expansion of newly activated cells.4 Immature thymocytes have also been demonstrated to express IL-2 during distinct stages of their intrathymic development. For example, IL-2-specific mRNA has been detected in murine thymoyctes as early as day 13 of embryogenesis5 and is also readily apparent in thymic tissue of neonatal mice.6 IL-2 exerts also an important effect on the differentiation of cells other than T cells. Signalling via IL-2 increases the immunoglobulin synthesis and J-chain transcription of B lymphocytes;7 potently activates the cytolytic activity of natural killer ŽNK. cells;8 induces lymphokine-activated killer cells;8 promotes proliferation, differentiation and cytolytic capacity of macrophages;9,10 enhances the antibodydependent tumoricidal activity of monocytes; and functions as a trophic factor for stroma cells of the thymic microenvironment.11 Moreover, IL-2 is critically important for the immune system in general as mice homozygous for a targeted disruption of the IL-2 locus display multiple irregularities in their response to self-antigens12 and to a range of different viral antigens.13,14 Similarly, impaired IL-2 secretion
Key words: interleukin-2 r transcription factors r T cells r monoallelic expression Q1999 Academic Press
Introduction EXTENSIVE COMMUNICATIONS between lymphocytes, antigen-presenting cells and stromal cells are required for a normal immune system to generate an antigen-specific response. This interaction is mediated concurrently by direct cell]cell contacts via a panoply of cell surface molecules and an array of soluble mediators, collectively called cytokines. Interleukin-2 ŽIL-2. is a prominent cytokine and acts as a major growth factor for the clonal expansion of T cells.1 Together with IL-4, IL-7, IL-9 and IL-15, IL-2
From the Pediatric Immunology Department of Research and The Children’s University Hospital, Hebelstrasse 20, 4031 Basel, Switzerland. E-mail: [email protected] Q1999 Academic Press 1044-5323r 99 r 050357q 11 $30.00r 0
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by activated T cells has been associated with functional immunological defects in man.15 ] 18 The importance of IL-2 in maintaining normal T cell homeostasis has been highlighted by the observation that IL-2 is a critical determinant involved in programmed cell death.19 In vivo, antigen receptor stimulation of mature ab TCRq lymphocytes previously exposed to IL-2 leads to apoptosis while the concomitant blockade of IL-2 with antibodies reverses this marked deletion. Thus, IL-2 may also participate in a feedback regulatory mechanism by predisposing mature T lymphocytes to apoptosis. In contrast, IL-2 overproduction results in the loss of T cell anergy in vitro 20 and causes the functional enhancement of self-reactive T cells in vivo leading to autoimmunity.21 Based on these numerous observations the conclusion may be drawn that IL-2 synthesis has to be tightly regulated in order to assure normal immune homeostasis. This contention is further reflected by the fact that IL-2 gene expression is regulated almost exclusively at the transcriptional level where three different but partially interrelated mechanisms are operational: Ži. the dynamic and cooperative assembly of diverse transcription factors; Žii. cell-type specific alterations in chromatin structure; and Žiii. a mechanism of monoallelic transcription. Combined, these diverse mechanisms guarantee that T cell lineage- and activation-specific IL-2 expression will occur in response to a variety of biochemical signals but in accordance with a discriminatory threshold.
torŽs. via a signaling pathway which is functional only in T cells. A third possibility assumes that activation dependency and cell-type specificity are mediated by two separate mechanisms: Transcriptional activation is induced in response to physiological stimuli Žwhich do not have to be T cell specific. but T cells have specifically altered their chromatin to accommodate for IL-2 gene accessibility. IL-2 regulation was first confined to a minimal IL-2 DNA regulatory sequence mapped to the first 300 base pairs Žbp. proximal to the start site.23 Further analysis characterized the organization of this transcriptional control element and defined extensive sequence homologies between mouse and man.24 Within this minimal IL-2 promoterrenhancer, several binding sites have been defined for tissue restricted and ubiquitously expressed transcription factors including NF-ATs, AP-1, NF-k B, and Oct-1, respectively Žreviewed in refs 22,25,26.. Some of these factors interact with their target DNA sequence without prior activation Že.g. Oct-1. while the majority of transcriptional activators implicated in IL-2 transcription associate with their DNA binding sites only after specific activation Že.g. NF-AT, AP-1, NF-k B..27 ] 29 Although these transcription factors allow for the induction of an IL-2 reporter gene in T-lymphocytes, none of these factors are lineage-specific. Moreover, the individual regulatory sequences within the IL-2 promoter are non-consensus sites that bind their cognate factors with an up to 10-fold lower affinity than the corresponding consensus sites.30 Importantly, mutations of each of these sites to the corresponding consensus sequences results in the loss of T cell transcriptional specificity demonstrating that the binding affinity of ubiquitous factors contributes Žat least in part. to the cell-type specific activity.30 To date, none of the factors necessary for IL-2 expression using the minimal enhancerrpromoter sequence has been demonstrated to be T cell-specific. However, this conclusion does not exclude the possibility that T cell lineage-specific protein]DNA interactions outside of the minimal promoterrenhancer account for ‘architectural’ chromatin changes which ‘sensitize’ the IL-2 locus to the binding of activationinduced transcription factors. The contention that long range interactions may be operational well beyond the positively regulating proximal DNA sequences has been put forth by the above results and by analysis of transgenic mice. The use of a construct containing the E. coli lac Z reporter gene under the control of a 583 bp promoterrenhancer sequence immediately upstream of
Regulatory sequence elements within the IL-2 gene The restriction of IL-2 expression to activated T cells provides evidence for a regulatory mechanism driven by two kinds of information: Ži. transient changes in the provision of biochemical signals activate factors which control IL-2 transcription; and Žii. lineagespecific determinants impose a tissue restriction for the control of IL-2 expression. Several possibilities have been suggested that bring these two mechanisms together and that allow for the IL-2 gene transcription to occur.22 One scenario predicts that IL-2 expression depends on at least one constituatively expressed transcription factor that is T cellspecific but has to interact with activation-induced factors in order to initiate IL-2 expression. A second model foresees that T cell-restricted IL-2 production is exclusively dependent on the activation of Ža. fac358
On the stochastic regulation of interleukin-2 transcription
the IL-2 start site was not sufficient to convey in vivo a position independent expression since only one of 17 transgenic mice expressed this reporter gene.31 Molecular investigations have thus not yet identified a moderately sized genomic sequence sufficient to regulate IL-2 gene expression in an inducible but lineage-restricted and integration independent manner. Despite this shortcoming, extended analysis of 2500 bp 59 of the minimal promoterrenhancer identified sequences which either intensify or suppress the magnitude of an inducible murine IL-2 response.24 Deletions within this distal cis-element yet reveal that no region of this sequence altered either the requirement for induction, the kinetics of stimulation, nor the cell-type specificity of IL-2 expression. Even in the presence of extensive 59 flanking sequences, reporter gene transcription remains strongly dependent on the factors binding within the first 300 bp upstream of the start site stressing the conclusion that the additional 59 sequences exert at most a secondary modulating effect on IL-2 transcription. The observation that optimal cytokine production, especially in naıve ¨ T cells, requires signaling via the TCR and engagement of co-receptors raises the possibility that only activated T cells generate a specific transcription factor which accounts for cell- and activation-restricted IL-2 transcription. ŽAlternatively, costimulatory signals may induce transcription by altering the molecular composition of transcription factors as has been shown for AP-1.32 . Engagement of the surface protein CD28 provides co-stimulatory signals critical for the integral activation of T cells and the subsequent production of IL-2. CD28-mediated signals exert their stimulatory effect via a sequence designated the CD28 response element ŽCD28RE..33,34 CD28RE constitutes a variant NF-k B site 35 and is adjacent to and functionally cooperates with an AP-1 binding sequence.36 These findings preclude the existence of a T cell-specific transcription factor that exclusively integrates CD28 Žor other co-receptor. mediated signals. Although tissue-specific repressors of IL-2 transcription have not yet been identified, several lines of evidence suggest that different DNA binding factors may act as negative regulators Že.g. p50 of the family of NF-k B molecules, ZEB, Ets-1 and others.. A mechanism of silencing could contribute in several ways to the documented restriction in IL-2 transcription: Ži. tissue specificity; Žii. silencing of IL-2 expression in fully differentiated Th2 cells; and Žiii. loss of IL-2 transcription in anergic T cells. Mutational analyses of the IL-2 promoterrenhancer have defined a re-
pressor element that overlaps with a negative regulatory element, designated NRE-A.37,38 Binding of the highly conserved ‘two-handed’ zinc finger protein ZEB to NRE-A has been implicated in the silencing of the IL-2 gene in activated Th2 cells.39 However, this repressor factor is not T cell-specific.40 Moreover, T cells rendered anergic display an altered composition of their transcription factors, which most likely effect IL-2 repression in anergic T cells. For example, in vivo anergized CD4q T cells have been demonstrated to display severely perturbed levels of AP-1 and FosrJun-containing NF-AT complexes and NF-k B complexes composed of the transcriptionally inactive p50 homodimer.41,42 The available data concerning the role of transcription factors governing IL-2 expression suggest that the cooperative binding of individual factors to weak sites within the promoterrenhancer renders the entire complex competent for lineage-specific and activation induced IL-2 transcription. A requirement for stereospecific contacts among distinct transcription factors themselves may be of additional importance. Moreover, in vivo footprinting has implied that nuclear factors fail to interact with the minimal promoterrenhancer in unstimulated cells and non-T cells,43 suggesting additional epigenetic features specific in activated T cells. Thus, gene transcription is not the mere sum of multiple independent transcription factors binding to their corresponding DNA sequence. Rather, transcriptional activation is determined by an architectural assembly of interactive nucleoproteins which form distinct protein]DNA and protein]protein complexes that execute a precise IL-2 regulation at the minimal promoterr enhancer.44
Regulation of IL-2 transcription by chromatin changes Histones, linker histones, high molecular group ŽHMG. molecules and other proteins package the eukaryotic DNA into distinct nucleosomal arrays. This organization of the genome into nucleoprotein complexes is essential for the expression and silencing of genetic material, and thus acts as a major regulator in development. Moreover, the exact position of a gene with regards to DNA packaging proteins influences the expression level as DNA binding proteins impart a different stability to the wrapping of DNA.45 Hence, chromatin architecture plays a key role in the events leading to transcription46 and alterations in the chro359
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matin structure have thus been proposed to govern the T cell-restricted22 and possibly allele-specific IL-2 expression. A direct approach to determine the organization of the nucleoprotein architecture is to use in vivo DNase I digestion of chromosomal DNA. This nuclease analysis can identify distortions in the tertiary structure of the DNA helix induced by protein binding. It is of note that DNase I hypersensitive sites are not necessarily histone-free but may actually inflect the recruitment of nuclease by transcription factors bound to an intact nucleosomal infrastructure.47 In vivo DNase I footprinting of sequences y600 to q400 relative to the IL-2 start site reveals the existence of several, tissue-specific and stimulation independent differences in the DNA structure.48 These hypersensitive sites ŽHS. are confined to a 59 sequence of the fragment analyzed. Importantly, these distinct HS of resting T cells do not repress IL-2 gene transcription as they are equally present in activated T cells. Tissue-specific DNA]protein interactions have also been directly established for a sequence centered around residue y410.48 This particular site is recognized by HMG proteins which, in turn, may cooperate with other factors present in the nucleus to form specific complexes. These proteins may alter the overall architecture of the 59 sequence with regard to the minimal promoterrenhancer located at nucleotides y300 to y45 relative to the IL-2 start site. The interaction of HMG or other nucleoproteins acting as ‘lineage’ specific, constitutive marking proteins with DNA sequences beyond residue y390 could ‘open’ the chromatin of the minimal promoterrenhancer and render this regulatory sequence sensitive to activation by transcription factors. Upon successful T cell stimulation, additional DNase I HS can be noticed within the region of the IL-2 promoterrenhancer revealing the induction of activation-induced changes in chromatin conformation.48 These latter alterations are Žmost likely. caused by the binding of transcription factors to their respective binding sites. Studies focusing on chromatin structure have not addressed yet the issue of position independence of the IL-2 regulatory sequence. Although in vivo footprinting has identified tissue specific chromatin features within 600 bp 59 to the start site, the use of an almost identical sequence as a transgene has not conveyed protection against position effects.31 Thus, the existence of genomic sequences further 59 may mark the boundary of the active chromatin domain and may function as an insulator. In this context, it
has recently been shown that mammalian insulator sequences can prevent the loss of transcription of a reporter gene, which usually adopts the epigenetic hallmarks of an inactive gene,49,50 i.e. nuclease inaccessibility, DNA hypermethylation and histone hypomethylation. Taken together, these descriptive studies of chromatin structure in vivo have so far delineated that the IL-2 gene adopts a T cell-lineage specific architecture which is further altered upon lymphocyte activation. These findings fulfill the general demand that celltype specific and induction-dependent chromatin changes regulate IL-2 transcription. Accordingly, chromatin remodeling of IL-4 and IL-13 genes has recently been demonstrated and these changes correlate with a differentiation to a Th2 effector phenotype.51
Monoallelic expression of IL-2 Monoallelic expression is an epigenetic phenomenon in which only a single copy, or allele, of a given gene is expressed. This is unusual in that most genes of diploid organisms are expressed equally from each copy Žwith X chromosome encoded genes constituting a notable exception.. Monoallelic gene transcription has recently been demonstrated as a third regulatory mechanism operational in the control of IL-2 transcription.52 Although immature thymocytes have an ‘open’ IL-2 locus and have been shown to transcribe IL-2,5 it is presently not known whether monoallelic expression is already fixed at this developmental stage or whether this mode of transcriptional regulation is established later. Initially it was observed that the bulk of peripheral T cells heterozygous for a null mutation of the IL-2 gene ŽIL-2qry . produced only half as much IL-2 when compared to wild type ŽIL-2qrq . T cell cultures. This result could be accounted for by two mutually exclusive models: First, each of the T cells in both cultures produce IL-2 but, when analyzed at a single cell level, a IL-2qry T cell synthesizes only half as much of the cytokine in contrast to a IL-2qrq T cell. Alternatively, the pool of IL-2qry T cells consists of two equally sized subgroups, that is one population that produces IL-2 and the other group that fails to do so. This latter model posits two testable properties: first, the frequency of IL-2 secreting cells among IL-2qry T lymphocytes has to be half of that noticed for wild type T cells, and second, IL-2 producing T cells bearing only one functional IL-2 allele produce iden360
On the stochastic regulation of interleukin-2 transcription
tical quantities of the cytokine per single cell when compared to T cells with two functional alleles. The analysis of mature thymocytes and peripheral T cells heterozygous for an IL-2 null mutation revealed in young mice that only approximately half of all the cells produced IL-2 upon stimulation.52 Moreover, single cell quantification of intracytolplasmic IL-2 by use of digital analysis with an interactive laser cell cytometer demonstrated that heterozygous but IL-2 synthesizing T cells did produce the cytokine as copiously as wild type T cells. The similar frequency of IL-2 producing versus IL-2 non-producing mature thymocytes and peripheral T cells in young heterozygous mice argues for a random process of silencing of one of the IL-2 loci and concurrently argues against a control feed-back mechanism which assures a preferential or even exclusive transcription from the functional allele. Moreover, these results also demonstrate that the autocrine use of IL-2 is not a decisive element for intrathymic T cell maturation. In contrast, analysis of older mice demonstrated that IL-2qry peripheral T cells competent to secrete IL-2 have a growth advantage over heterozygous cells which fail to express this cytokine secondary to silencing of their functional allele.52 This result implies that the silencing of one of the two IL-2 alleles is stable for each clonal progeny and thus does not appear to be ‘negotiated’ anew with every cell division. ŽHowever, formal proof of this assertion is presently not available because ‘allelic fidelity’ of IL-2 silencing has not yet been reported at the clonal level.. Considering the situation of wild type ŽIL-2qrq . T cells, the con-
clusion that IL-2 is regulated in an allele-specific manner is further underscored by single cell RT-PCR analysis. Single T cells with allele-specific IL-2 sequence differences express usually either the maternal or the paternal allele but not both. Taken together, these cellular and molecular analyses in primary murine T cells argue for a monoallelic control of IL-2 transcription. The monoallelic transcription of the IL-2 locus was recently questioned in an experiment using transgenic mice where the first exon of the IL-2 gene was replaced with cDNA encoding the green fluorescent protein ŽGFP..53 Upon activation of T lymphocytes heterozygous for this ‘knock-in’, concomitant expression of IL-2 and GFP is readily visible in approximately 60% of single cells analyzed. Among the remaining T cells a majority expresses exclusively IL-2 while a small fraction of cells is positive only for GFP. This finding is difficult to reconcile with monoallelic expression of IL-2 as long as the molecular mechanisms regulating transcription from only one of the two alleles is not determined. However, the loss of exon 1 of the IL-2 locus, the introduction of GFP cDNA and the insertion of polyadenylation sequences may account for the disturbance of regulatory elements by either removal or the inclusion of regulatory sequences.
Monoallelic expression of other cytokines Two independent experimental approaches recently
Table 1. Characteristics of monoallelic gene expression Žn.d.s not determined. Gene
Replicative asynchrony
Time of establishment
X-linked genes in females
Yes
Early in embryogenesis
Imprinted genes
Yes
T and B cell receptor genes
Parental origin of silent allele
Allelic difference in chromatin structure or factor binding
Requirement for functional gene product Žfeed back.
Random
Yes
No
Gametogenesis
Non-random
Yes
No
n.d.
Lymphopoiesis
Random
Yes
Yes
NK cell receptor genes
n.d.
n.d.
Random
n.d.
n.d.
Olfactory genes
Yes
Early in embryogenesis
Random
n.d.
n.d.
IL-2 gene
Yes
n.d.
Random
n.d.
No
361
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documented that IL-4 transcription is also controlled in an allele-specific manner. Analysing activated wild type T cells biased for a Th2 phenotype and bearing two IL-4 alleles which allow assignment of the parental origin revealed that each of the two alleles is regulated independently with transcription occurring preferentially from a single allele.54 The second model took advantage of a mutant mouse in which one of the IL-4 alleles was replaced by the human CD2 cell surface molecule.55 Upon stimulation, most of the cells from these transgenic mice expressed only IL-4 or the CD2 reporter gene, thus revealing the stochastic nature of IL-4 transcription. Interestingly, the increased probability of allelic transcription Žand thus the loss of mono-allelic expression. was directly correlated to the strength of the activation signal delivered via the TcR. In addition to IL-4, also the transcription of GM-CSF appears to be regulated via a stochastic process.54 Thus, allele-specific activation may reflect a general feature of cytokine transcriptional regulation.
Interestingly, X inactivation is mediated by a posttranscriptional mechanism, i.e. the stabilization of Xist RNA, rather than by the regulation of the Xist promoter. Imprinting reflects the silencing of an allele in a heritable and parental-specific manner.64 Established already during gametogenesis, imprinted genes are frequently clustered within the genome and are associated with asynchronous replication during S-phase of the cell cycle Žreviewed in ref 65.. From the onset, imprinting has been associated with differential DNA methylation for most but not all of the genes.66 However, it remains to be determined whether the role of methylation is to act as the gametic imprinting signal or whether it merely maintains allele-specific expression. Interestingly, in some instances, imprinting is restricted to only certain organs suggesting the existence of tissue-specific transacting factorŽs..67 Experimental evidence proposes several additional mechanisms to be operational for imprinting to occur including the existence of chromatin insulators; transcriptional and post-transcriptional interference with anti-sense RNA; and enhancer competition. In each of these models, allele-specific DNA methylation is either directly or indirectly responsible for gene silencing. The genes for the antigen receptors of B and T lymphocytes undergo recombinations during distinct stages of maturation. The productive chromosomal rearrangement of one allele yields a functional receptor chain which, using a feed-back mechanism, precludes the second allele to undergo chromosomal rearrangement.68 Trans-acting proteins, enhancers and cis-regulatory sequences mediate local changes in chromatin structure rendering the loci of the differ ent antigen receptor chains accessible to the recombination apparatus. In vitro experiments have demonstrated that DNA methylation inhibits69 and that undermethylation is required for the rearrangement of B and T cell antigen receptor genes.70 These findings are compatible with the hypothesis that demethylation directs the initial choice of a single allele for rearrangement. The family of murine Ly49 NK cell receptors are MHC class I specific antigen receptors belonging to the C-type lectin superfamily. Monoallelic expression of these receptors generates a diverse repertoire of NK cells with restricted specificities as single NK cell receptors recognize different MHC class I molecules.71 Expression of these receptors in mice heterozygous for the Ly49A, Ly49C and Ly49G2 gene is usually effected from one or the other allele and
Comparative characteristics of monoallelic gene expression In addition to the IL-2 and IL-4 genes, other examples of allele-specific expression include X-inactivation,56 parental imprinting,57 the monoallelic exclusion of the antigen receptors of T and B lymphocytes and the stochastic selection of both natural killer ŽNK. 58,59 and olfactory receptors.60 Although bearing some common features, the mechanisms responsible for these forms of monoallelic gene expression appear to be distinct from the characteristics of the IL-2 locus Žsee Table 1.. X-inactivation assures the correct gene dosage for cells of different gender so that female cells will express the same number of X-linked genes as male cells. This phenomenon of gene dosage compensation results from mechanisms that first count the chromosome number and then inactivate the supernumerous X-chromosomeŽs..60,61 The counting mechanism allows for only one of the X-chromosomes to be active and this task may be achieved randomly by a trans-activating ‘blocking’-factorŽs.. The Xist gene, which maps to the X chromosome inactivation center ŽXic. and encodes an untranslated RNA, is required for initiation, propagation and maintenance of the inactivation process.62 A process of DNA methylation is responsible for the silencing of the Xist gene on the inactive X-chromosome.63 362
On the stochastic regulation of interleukin-2 transcription
only rarely from both loci.58,59 Monoallelic Ly49 gene expression appears to arise as a consequence of a stochastic Ly49 gene activation mechanism although the molecular details responsible for it have not yet been elucidated. The olfactory receptor gene family contains approximately 1000 genes, which are clustered within multiple loci that are broadly distributed in the genome. These loci lie within paralogous chromosomal regions that appear to have arisen by duplications of large chromosomal domains followed by extensive gene duplication and divergence.72 In individual olfactory neurons, the expression of a given receptor derives exclusively from one allele and only one allelic array encoding multiple receptor genes is active.73 Alleles encoding the odorant receptors are replicated asynchronously, further emphasizing the phenomenon of allelic inactivation. This random inactivation of one allelic array and the concomitant cis-control of the active allele are established early in development and may also affect cells of non-neuronal lineage.73 A cis stochastic model of olfactory gene regulation has been proposed to control first allelic inactivation which is then followed by mechanisms that chose first one of the several chromosomal arrays on a single allele and then a single receptor.73 The precise molecular mechanisms operational in this allele-specific expression are, however, not known to date. Expression of the IL-2 gene is}as already alluded to}in many aspects different when compared to the examples outlined above: Ži. IL-2 is present as a singular copy located on mouse chromosome 3 Žposition 19.2.;74 Žii. this locus is not known to be imprinted;75 Žiii. IL-2 expression is independent of recombination;76 and Živ. there is no feed-back mechanism operational which ensures that the expression from a defective allele encoding IL-2 is switched to the functional allele providing a normal gene product.52
lated by at least two conceptually different mechanisms. One model could predict that a single IL-2 allele is randomly marked Žfor example by differential methylation. and consequently silenced by a molecular mechanism either similar or identical to those modeled for the phenomenon of imprinting. Alternatively, transcription of IL-2 may be achieved by a limited concentration of specific transcription factors, which allows at any given time for the occupancy of only one of the two promoters of the IL-2 locus. The inverse correlation between DNA methylation and tissue-specific gene expression has been established for in vivo transcription.77 The covalent modification of cytosine to methyl-cytosine may change the chromatin structure and consequently the binding of the transcriptional machinery to the double helix.78,79 Such a structural modification controls transcriptional gene activity either at a local Ži.e. monogenetic. level or, alternatively, may influence many genes within a chromosomal stretch of variable length. The correlation between demethylation and activation of genetic loci has been observed for several genes in the immune system,80 including the IL-4 locus. This latter example is relevant to the discussion here because the IL-4 locus is hypermethylated in naıve ¨ T cells but not in polarized Th2 cells81 where IL-4 expression occurs frequently in a monoallelic manner.54,55 Following initial stimulation and demethylation, the ensuing chromatin remodeling of the IL-4 locus proceeds Žor at least parallels. transcriptional activity.82,51 This change to an ‘open’ chromatin configuration Žnow responsive to transcriptional activation. may very well correspond to the structural alterations seen in the IL-2 locus of activated T cells.48 Whether demethylation andror chromatin changes occur selectively for only one of the two alleles of activated primary T cells has not yet been directly evaluated. Circumstantial evidence suggests, however, that there is a difference in chromatin structure between the active and the silent IL-2 allele as asynchronous DNA replication has been reported for this locus in dividing primary T cells.52 Asynchrony of replication has previously been shown for imprinted and X chromosome inactivated genes but not for most other genes with the notable exception of the olfactory gene.83 ] 86 Although an allelespecific difference in methylation has not yet been reported for the IL-2 locus, differential methylation has been documented where the silenced allele is methylated while the expressed allele of an imprinted gene is not.67,87 Marking by differential methylation
Models for monoallelic IL-2 expression The precise molecular mechanisms regulating monoallelic IL-2 expression are presently unknown. However, any model explaining monoallelic IL-2 transcription not only needs to clarify the stochastic nature of this process but has to accommodate the particular characteristics of the IL-2 locus and its transcriptional activation restricted to T cell stimulation. Basically, monoallelic expression may be regu363
G. A. Hollander ¨
andror the presence of a specific DNA binding protein affecting only one of the two alleles may be the discriminatory process operational in the monoallelic control of the IL-2 locus. A second model explaining monoallelic transcription of the IL-2 locus could foresee that allele-specific transcription is regulated probabilistically by the correct combination of transcription factors. Although similar to the mode of regulation of most genes, this model stipulates that the stochastic activation of only one of the two alleles is entirely dependent on a limited concentration of the transactivating complex. Thus, at any given time during IL-2 transcription and possibly largely independent of the degree of a stimulus, the factors available will only be sufficient to transactivate one of the two promoters. Disregarding physical constraints related to the positioning of the alleles within the nuclear architectural organization, an extreme prediction of this model would then anticipate that a single transcription complex is sufficient to initiate transcription in an activated T cell. In general terms, this second model also provides the mechanistic basis for unstable gene expression, a genetic pattern where a particular locus is transcribed only intermittently and preferentially from a single allele Žreviewed in ref 88]90.. Observations in cloned T cells, where the initial activation of an IL-2 reporter construct represents a probabilistic event, provide experimental support for such a second model.91 There are, however, several findings regarding IL-2 transcription, which are difficult to reconcile with this second model. Foremost, analysis of single F1 T cells bearing two IL-2 alleles separable by parentalspecific sequences have not revealed within 10 h after stimulation a bi-allelic expression at the RNA level. Normally, IL-2 transcripts can be detected as early as 2 h after mitogenic activation of primary resting T cells. They are at their highest levels between 4 and 8 h after stimulation and decline subsequently to be completely absent by 24 h.53,92 These results imply that if unstable IL-2 gene expression were to occur, the transcriptional machinery would have to occupy a single allele for an extended period of time Ži.e. ) 8 h.. There is also an alternative explanation for this result: if the half-life of the IL-2 specific message were extremely small}that is considerably shorter than the reaction kinetics of transcription itself}a comparable outcome would be anticipated. The observed rate of increase of IL-2 transcripts in activated T cells invalidates, however, this explanation92 if one does not postulate at the same time that the transcriptio-
nal frequency of a single allele is considerably higher within the first 4]8 h after stimulation when compared to earlier or later time points. An other result is also not apparently compatible with a model of slow-unstable gene expression from a single functional allele.90 The digital quantitation of intracytoplasmic IL-2 in activated T cells from IL-2qrq and IL-2qry mice reveals the same range of cytokine concentrations per single cell. Finally, T cells from old mice heterozygous for a null mutation reveal an obvious increase in the frequency of IL-2 expressing cells 52 which suggests a fixed allelic pattern of transcription Žas described for IL-4 producing clones54 .. Therefore, any mechanism regulating monoallelic transcription of cytokines will need to explain the symmetric segregation to the sister chromatids during cell division. A model of limited availability of transcription factors results in a random choice of a single allele but fails to easily explain an allelic pattern of IL-2 expression that is transmitted as a stable epigenetic trait
Conclusion The purpose of cytokines being expressed in a monoallelic manner remains at present ill-explained but is certainly of teleologic interest. For the IL-4 locus it was argued that a probabilistic expression of only one allele allows a combinatorial assortment of distinct cytokine genes among the clonal progeny of T cells with an identical antigen receptor specificity.54 There is still another evolutionary explanation to be considered. Avoiding transcription from both IL-2 alleles, the organism takes advantage of diploidy. Such a restriction to monoallelic usage may bear the benefit that IL-2 isoforms can be functionally sampled during an immune response as different IL-2 sequences have been described encoding variable biological activities.93 Consequently, single T cells expressing a biologically superior isoform will have a growth advantage which ultimately benefits the entire individual. A monoallelic mechanism will also diminish the representation of serious recessive mutations at the clonal level and may thus be decisive for the outcome of an immune response. However, critical for any attempt of an explanation is the issue whether a twofold difference in the in vivo expression of a cytokine has a functional impact on a system as complex as the immune system. Results obtained in an allogenic transplantation model support the in vivo importance of such a relatively small variance in 364
On the stochastic regulation of interleukin-2 transcription
12. Sadlack B, Merz H, Schorle H, Schimpl A, Horak I Ž1993. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75:253]261 13. Cousens LP, Orange JS, Biron CA Ž1995. Endogenous IL-2 contributes to T cell expansion and IFN-gamma production during lymphocytic choriomeningitis virus infection. J Immunol 155:5690]5699 14. Kundig TM, Schorle H, Bachmann MF, Hengartner H, Zinkernagel RM, Horak I Ž1993. Immune responses in interleukin-2-deficient mice. Science 262:1059]1061 15. Chatila T, Castigli E, Pahwa R, Pahwa S, Chirmule N, Oyaizu N, Good RA, Geha RS Ž1990. Primary combined immunodeficiency resulting from defective transcription of multiple T-cell lymphokine genes. Proc Natl Acad Sci USA 87: 10033]10037 16. Doi S, Saiki O, Tanaka T, Ha-Kawa K, Igarashi T, Fujita T, Taniguchi T, Kishimoto S Ž1988. Cellular and genetic analyses of IL-2 production and IL-2 receptor expression in a patient with familial T-cell-dominant immunodeficiency. Clin Immunol Immunopathol 46:24]36 17. Pahwa R, Chatila T, Pahwa S, Paradise C, Day NK, Geha R, Schwartz SA, Slade H, Oyaizu N, Good RA Ž1989. Recombinant interleukin 2 therapy in severe combined immunodeficiency disease. Proc Natl Acad Sci USA 86:5069]5073 18. Weinberg K, Parkman R Ž1990. Severe combined immunodeficiency due to a specific defect in the production of interleukin-2. N Engl J Med 322:1718]1723 19. Lenardo MJ Ž1991. Interleukin-2 programs mouse alpha beta T lymphocytes for apoptosis. Nature 353:858]861 20. Essery G, Feldmann M, Lamb JR Ž1988. Interleukin-2 can prevent and reverese antigen-induced unresponsiveness in cloned human T lymphocytes. Immunology 64:413]417 21. Andreu-Sanchez JL, Alboran I, Marcos MAR, Sanchez-Movilla A, Martinez-A C, Kroemer G Ž1991. Interleukin-2 abrogates the nonresponsive state of T cells expressing a forbidden T cell receptor repertoire and induces autoimmune disease in neonatally thymectomized mice. J Exp Med 173:1323]1329 22. Rothenberg EV, Ward SB Ž1996. A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin 2 gene regulation. Proc Natl Acad Sci USA 93:9358]9365 23. Fujita T, Shibuya H, Ohashi T, Yamanishi K, Taniguchi T Ž1986. Regulation of human interleukin-2 gene: functional DNA sequences in the 59 flanking region for the gene expression in activated T lymphocytes. Cell 46:401]405 24. Novak TJ, White PM, Rothenberg EV Ž1990. Regulatory anatomy of the murine interleukin-2 gene. Nucleic Acids Res 18:4523]4533 25. Jain J, Loh C, Rao A Ž1995. Transcriptional regulation of the IL-2 gene. Curr Opin Immunol 7:333]342 26. Serfling E, Avots A, Neumann M Ž1995. The architecture of the interleukin-2 promoter: a reflection of T lymphocyte activation. Biochim Biophys Acta 1263:181]200 27. Serfling E, Barthelmas R, Pfeuffer I, Schenk B, Zarius S, Swoboda R, Mercurio F, Karin M Ž1989. Ubiquitous and lymphocyte-specific factors are involved in the induction of the mouse interleukin 2 gene in T lymphocytes. Embo J 8:465]473 28. Brunvand MW, Schmidt A, Siebenlist U Ž1988. Nuclear factors interacting with the mitogen-responsive regulatory region of the interleukin-2 gene. J Biol Chem 263:18904]18910 29. Rooney JW, Sun YL, Glimcher LH, Hoey T Ž1995. Novel NFAT sites that mediate activation of the interleukin-2 promoter in response to T-cell receptor stimulation. Mol Cell Biol 15:6299]6310 30. Hentsch B, Mouzaki A, Pfeuffer I, Rungger D, Serfling E Ž1992. The weak, fine-tuned binding of ubiquitous transcription factors to the Il-2 enhancer contributes to its T cell-restricted activity. Nucleic Acids Res 20:2657]2665
IL-2 production. Analysing Graft-versus-Host disease ŽGVHD. in lethally irradiated mice reconstituted with both allogenic bone marrow cells and T cells bearing two or one functional IL-2 allele, survival could be directly correlated to the IL-2 gene dosage ŽG. Hollander, manuscript in preparation.. These results ¨ demonstrate that a two-fold difference in IL-2 expression has a definite bearing on the outcome of an immune response. This is not entirely unexpected as relatively small concentration gradients of growth and differentiation factors constitute a central principle in developmental biology. The elucidation of the molecular mechanisms governing allele-specific IL-2 transcription becomes therefore the more important.
Acknowledgements I would like to thank E. Palmer and N. Iscove for helpful discussions and review of the manuscript. This work was supported by grants from the Swiss National Science Foundation Ž31’43600.95 and 3239-055233.98r1..
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