Interleukin 7 Receptor Control of  T Cell Receptor γ Gene Rearrangement: Role of Receptor-associated Chains and Locus Accessibility

Embryonic thymus was obtained by performing timed breeding of C57BL/6 mice maintained at Animal Production (Frederick, MD). IL-7Rα^−/− mice (1) and Rag2^−/− mice (36) were produced in the NCI facility (Frederick, MD) from breeders purchased from The Jackson Laboratory (Bar Harbor, ME). γ_c^−/− mice (37) were bred in the National Institutes of Health (NIH) facility (Bethesda, MD). Jak3^−/− mice (38) were bred at the University of Massachusetts Medical Center (Worcester, MA). Animal care was provided in accordance with the procedures outlined in the “Guide for the Care and Use of Laboratory Animals” (NIH Publication No. 86-23, 1985).

DNA was extracted (39) from adult thymi of the indicated strain or fetal C57Bl/6 thymus from day 15 of gestation. In the case of fetal Rag2^−/− or IL-7Rα^−/−, several thymi were pooled, whereas all other thymi were individually treated. Approximately 1 μg of DNA was used for each PCR reaction in 50 μl reaction buffer (PE Applied Biosystems, Inc., Foster City, CA) with 0.2 mM of each NTP, 1 μM of each primer, and 1 U of Taq polymerase. The PCR reaction was performed with a hot start: (94°C for 3 min) 94°C for 1 min, 55°C for 1 min, 72°C for 1 min for 30 cycles (extension, 10 min at 72°C). RNA was extracted with RNAzol according to the manufacturer's protocol and reverse transcribed as described with oligo(dT) primers (9). PCR was performed as for genomic DNA with the exemption that the annealing time was 2 min at 55°C. PCR products were separated on a 1.4% agarose gel, blotted, and pre-hybridized (Nycomed Amersham, Arlington Heights, IL) for 1 h before addition of the γ-ATP 5′ end-labeled probe. After 12–16 h of hybridization at 42°C, blots were washed according to the manufacturer's protocol (Nycomed Amersham) and visualized by autoradiography. In Fig. 3 (see Results), the PCR product has not been blotted, but rather is the negative image of an original ethidium bromide–stained agarose gel. The location of primers within the TCR-γ locus are shown in Fig. 1 (see Results). The primers for detection of DNA gene rearrangement were derived from sequences as previously published (40). The primers for detection of sterile transcripts have been published previously (41), as have the primers for detection of GAPDH mRNA (42). The following oligonucleotides were used (the bp number in parentheses indicates the precise position in the locus as reported in EMBL/GenBank/DDBJ under accession No. AF037352). No. 1, Vγ2 leader peptide sense (8,280 bp): 5′-CTGGGAATTCAACCTGGCAGATG-3′; No. 2, Vγ2 sense (8,460 bp): 5′-CATGGGAAGTTGGAGCAACCTGAAATATC-3′; No. 3, Vγ2 antisense (8,734 bp): 5′-GCTTCGTCTTCTTCCTCCAAGGAATA-3′; No. 4, Vγ2-3a′antisense (8,846 bp): 5′-GCTAAGAAGGATGTGGGTTG-3′, probe for Vγ2 (8,586 bp): 5′-AACCAAGGTTTAGAGTTTCTATTA-3′; No. 5, Vγ3 leader peptide sense (15,508 bp): 5′-CCAGCAGCCACTAAAATGTC-3′; No. 6, Vγ3 sense (15,678 bp): 5′-GACTCCTGGATATCTCAGGATCAGC-3′; No. 7, Vγ3 antisense (15,947 bp): 5′-CGTGGCTTCATCCGATGTGACAAC-3′; No. 8, Vγ3-3a′ antisense (16,059 bp): 5′-TGGAGGATCCTTGGTGGGTTCA-3′, probe for Vγ3 (15,795 bp): 5′-CTGAAAGAAGGGGAGCCCCTGAGACG-3′; No. 9, Jγ1 antisense (33,661 bp): 5′-CGGGATCCCAGAGGGAATTACTATGAGC-3′; No. 10, TCR-γ constant first exon sense (37,363 bp): 5′-AGGCTTGATGCAGACATTTCCCCC-3′; No. 11, TCR-γ constant first exon antisense (37,657 bp): 5′-GATCTGCTCCTCCTTTGTTGTTCTC-3′; No. 12, TCR-γ constant second exon antisense (39,154 bp): 5′-CTTGCCAGCAAGTTGTAGGCTTGG-3′, probe for TCR-γ constant (37,504 bp): 5′-GATGGCAATACTATCCTGGACTCC-3′.

Nuclear extracts were prepared (43) from ∼10⁷ fetal thymocytes (day 15 of gestation) from normal C57BL/6 mice, or from adult mice (4–6-wk-old) of Rag2^−/− or IL7Rα^−/− strains. The binding reaction was conducted at room temperature for 20 min with 1 to 3 μg of protein in 25 μl of binding buffer (25 mM hepes, pH 7.5, 10% glycerol, 1 μg poly[d(I-C)], 50 mM NaCl, 0.05% NP-40, 1 mM dithiothreitol) and 10,000 cpm of γ-[³²P]ATP 5′ end-labeled oligonucleotide. The reaction mixture was separated on a 7% polyacrylamide gel at 4°C, dried, and visualized by autoradiography. The nuclear extracts were tested for equal binding to the control nucleotide of the housekeeping factor Sp-1. For competitive inhibition of the gel shift, 50-fold excess of the specific oligonucleotide or a nonspecific competitor was used (the nuclear factor [NF]γ2 site for binding to NFγ3 or NFγ4, and Sp-1 for binding to NFγ2). For inhibition with antibodies, the extracts were incubated with 1 μl of specific antisera against Stat5a and Stat5b (R&D Systems, Minneapolis, MN) or control rabbit serum at 8°C for 2 h before the addition of the radiolabeled oligonucleotides. The following double-stranded oligonucleotides were used for detection of nuclear proteins that bind to the murine TCR-γ enhancer (52). Nfγ3: 5′-CATAGGAGCAGTTAAACCACAGCCAG-3′, 5′-CTGGCTGTGGTTTAACTGCTCCTATG-3′; Nfγ2: 5′-GTTGCTTCCTGGAAAATGGTTAAAG-3′, 5′-CTTTAACCATTTTCCAGGAAGCAAC-3′; and Nfγ4: 5′-CTTTCGAAAGACCACAGCTATTAG-3′, 5′-CTAATAGCTGTGGTCTTTCGAAAG-3′.

To analyze the methylation status of the TCR-γ locus, 15 μg of DNA was digested overnight with MspI or HpaII (which cannot cleave at methylated residues). The product was fractionated on a 0.8% agarose gel, blotted, and then prehybridized overnight. The blot was hybridized with a 294-bp probe generated by PCR from the first exon of the constant region of TCR-γ1 using primer Nos. 10 and 11 (as described above; see Fig. 1). The probe was random primed, then hybridized with the blot overnight in hybridization buffer containing 5× SSC, 10× Denhardt's solution, salmon sperm DNA (0.5 mg/ml), and 1% SDS. Blots were then washed 10 min in 3× SSC and 0.5% SDS at room temperature followed by washing 45 min in 0.1% SSC and 0.5% SDS at 60°C. The location of the MspI/HpaII sites in the TCR-γ locus is shown in Fig. 6.

Embryonic thymic lobes were obtained from C57Bl/6 embryos at day 14 of gestation. Lobes were depleted of endogenous thymocytes by irradiation (30 Gy), then reconstituted by placing in hanging drops of 35 μl of medium together with IL-7R^−/− bone marrow cells (5 × 10⁴/lobe) for 24 h. Reconstituted lobes were then washed and cultured in microtiter wells, one lobe per well, in 50 μl of medium for 96 h together with the specific histone deacetylase inhibitor TSA (Wako Chemicals).

It was previously shown that IL-7Rα^−/− thymocytes from two independent lines were defective in rearrangement of the TCR-γ locus (13, 14). A second chain, γ_c, serves as part of the receptor for IL-7 (26, 27), whereas it has been speculated that TSLP (25), a homologue of IL-7, signals through IL-7Rα together with a different chain. We therefore tested whether γ_c was required for rearrangement of the TCR-γ locus. Several rearrangements of this locus were examined using PCR, which generates a product if the locus is rearranged, bringing two gene segments in sufficiently close proximity to permit the polymerization reaction (Fig. 1). Using this method, thymocytes from various strains of mice were analyzed for rearrangement of Vγ3 and Vγ2, as shown in Fig. 2, and Vγ4 (data not shown), which showed the same pattern. As shown in Fig. 2, γ_c^−/− thymocytes were as deficient as IL-7Rα^−/− thymocytes in the rearrangement of this locus. Rag2^−/− thymocytes are shown as a control since they are unable to initiate VDJ recombination at any locus. We conclude that both IL-7Rα and γ_c chains are essential parts of the receptor complex that signals the rearrangement of the TCR-γ locus.

Several kinases are activated by IL-7R cross-linking, including those of the src (28, 30) and Janus families (29). One of the Janus kinases, Jak3, was examined for a possible role in signaling TCR-γ locus rearrangement because Jak3 is physically associated with γ_c and has been shown to be essential for normal lymphoid development (38, 44, 45). As shown in Fig. 3, a deficiency in TCR-γ rearrangement was noted in these Jak3^−/− mice that was comparable to the deficiency observed in thymocytes deficient in either IL-7Rα or γ_c chains. Thus, Jak3 is an essential component of the signaling pathway leading from IL-7R cross-linking to rearrangement of the TCR-γ locus.

Expression of Rag genes was previously shown to be sustained by IL-7 (9, 18, 46, 47) and it has been observed previously that transcripts for RAG1 and -2 are deficient in IL-7R^−/− thymocytes (10). We examined IL-7Rα^−/− thymocytes for expression of Rag1 and -2 and observed considerable variability (data not shown): different batches of thymocytes (pooled from five individuals) showed levels ranging from barely detectable to normal levels for both RAG1 and -2. Some IL-7Rα^−/− mice have been shown to display a “leaky” phenotype in that they develop small numbers of CD4⁺CD8⁺ cells (2); this cell population has been shown to express Rag messages in IL-7Rα^−/− thymocytes (10), whereas the same study observed suppressed Rag expression in the pro-T cells from the same mice. However, in our IL-7Rα^−/− mouse colony, some pools of thymocytes from “non-leaky” mice (with minimal numbers of CD4⁺CD8⁺ cells) also expressed high levels of Rag messages, so we presume that even among pro-T1 cells (CD44⁺CD25⁻) there is variability among individual mice. Thus, the deficiency in rearrangement of the TCR-γ locus, which is observed in all our IL-7R^−/− mice, could not be explained purely by a deficiency in expression of Rag genes. Moreover, rearrangement of the TCR-γ locus is more repressed than the other loci that undergo VDJ recombination (14), strongly suggesting that locus-specific influences are signaled by the IL-7Rα (5). We therefore sought additional mechanisms that could be involved in the severe repression of rearrangement of the TCR-γ locus in these mice.

A mechanism that is thought to control VDJ recombination is whether or not the locus is accessible to the RAG proteins (for review see reference 16). One indicator that the chromatin is open around a rearranging gene is the production of sterile transcripts emanating from that locus before its rearrangement, a phenomenon that has been reported for most of the loci that undergo VDJ recombination (48, 49), including the TCR-γ locus (50). Sterile transcripts from the TCR-γ locus were greatly reduced in IL-7Rα^−/− thymocytes as shown in Fig. 4. Transcription from both constant and variable regions were affected, and several different regions of the locus were repressed to a similar degree. Positive controls for production of sterile transcripts are day 15 embryonic thymus and RAG2^−/− thymus, both of which contain a high proportion of pro-T cells. The deficiency in sterile transcripts in IL-7Rα^−/− thymocytes suggests that signals from the IL-7R may control the accessibility of the TCR-γ locus, which would in turn affect its ability to be cleaved by the RAG proteins. However, an alternative explanation is that the locus is open before the IL-7R signal, which then induces its transcription, for example by inducing transcription factors that bind the enhancer and upregulate expression of the gene. We therefore performed additional assays to distinguish whether the transcriptional defect in IL-7Rα^−/− thymocytes was due to a lack of transcription factors or to inaccessibility of chromatin to those transcription factors.

To test for defects in transcription factors in IL-7Rα^−/− thymocytes, we examined nuclear proteins that bind to the enhancer regions which are located 3′ to three of the four constant regions of the TCR-γ locus. The enhancer confers lymphoid-specific transcription if coupled to a heterologous promoter (51, 52). Within the enhancer, three sites (NFγ2, 3, and 4) have been identified based on footprinting studies. The NFγ2 site resembles the consensus motif for binding of STAT5, which has been previously shown to be activated by IL-7 (29, 53). The proteins binding the NFγ3 site were shown to contain c myb and core-binding factor (54). The components of the complex that bind the NFγ4 site have not been identified. In Fig. 5, nuclear extracts from IL-7Rα^−/− thymocytes are compared with those from RAG2^−/− thymocytes, since thymic development in both of these strains is arrested at pro-T cell stages. The IL-7Rα^−/− thymocytes did not show a deficiency of complexes that bound any of the three enhancer sites. In some nuclear extract preparations from IL-7Rα^−/− thymocytes, an additional band was observed that migrated faster than the band in the control RAG2^−/− extract; we do not know whether this smaller complex is functionally significant, but suspect it represents a degradation product. In any case, we observed no clear deficiency in any complex in IL-7Rα^−/− thymocytes. Since STAT5 is activated by IL-7, and since the NFγ2 site resembles a STAT5 consensus site (see Materials and Methods for sequence), we tested whether the complex binding to that site contained STAT5a or -b using antibodies directed against these proteins. No effect of these antibodies was observed on the complexes, nor was complex formation blocked, nor was an increase in mass observed (as a positive control for the antisera, they were shown to supershift the STAT5 complex induced in the YT line by IL-2 stimulation; data not shown). Hence, the complex that binds the NFγ2 site does not appear to contain STAT5. In conclusion, we found no evidence that IL-7Rα^−/− thymocytes lacked any of the nuclear proteins previously implicated in activating the TCR-γ enhancer. However, some transcription factors can bind their motif in an “inactive” state, then phosphorylation allows them to interact with the transcriptional machinery (AP-1 is an example of such a transcription factor). Thus, it is possible that one of these three transcriptional complexes, although it binds DNA, is inactive in these thymocytes. It is also possible, since these sites were identified in lines of mature T cells, that pro-T cells use different sites to regulate transcription.

To determine whether signals from the IL-7R could control chromatin structure of the TCR-γ locus, we examined the methylation of this gene. Methylation of the cytosine residues in the sequence 5′ CpG are often associated with silencing of genes (for review see reference 55). This silencing is thought to be based on the binding of proteins to these methylated sites, thereby altering the access of transcriptional machinery (see below). Methylation is also able to block VDJ recombination of minichromosomes after replication (56). We previously noted that during T cell development, the TCR-β locus was demethylated just before undergoing VDJ recombination (Muegge, K., unpublished data). We therefore tested whether IL-7R signals controlled the methylation of the TCR-γ locus. This was indeed the case as shown by Southern blot analysis in Fig. 6. This assay is based on the properties of two restriction enzymes, MspI and HpaII, both of which cleave at the same restriction site, whereas methylation of the cytosine in that site interferes with cleavage by HpaII but not MspI. As shown, HpaII was unable to cleave sites in the TCR-γ locus in thymocytes from IL-7Rα^−/− or γ_c^−/−, whereas thymocytes from RAG2^−/− or SCID mice were cleaved by HpaII. Fig. 6 also illustrates the position of the methylation-sensitive cleavage sites within TCR-γ clusters 1 and 2 (the probe used for hybridization is 100% identical with cluster 1 and 95% identical with cluster 2). One site in cluster 1 generating the 7.7-kb fragment is located between the joining region and the constant region, whereas the other site is located within the first enhancer. This indicates that signals from the IL-7R induce demethylation of the TCR-γ locus, which may in turn keep the locus “open” for either transcription (production of sterile transcripts) or rearrangement. We have also examined the methylation status of the TCR-β locus (data not shown) and found that, in distinction to the TCR-γ locus, it appears to be equally demethylated in IL-7Rα^−/− and Rag2^−/− thymocytes. Thus, the requirement for IL-7R signals in rearrangement of the TCR-γ locus is consistent with an effect on accessibility.

Methylation of DNA is thought to obstruct accessibility by altering chromatin structure. The methyl-CpG binding protein MeCP2 interacts specifically with methylated DNA and mediates repression of transcription. It has been shown that MeCP2 complexes with transcriptional repressors including mSin3A and histone deacetylases (57). The specific histone deacetylase inhibitor TSA can relieve repressed transcription, indicating that histone acetylation is an important feature of accessible chromatin (57, 58). Thus, we tested whether the need for IL-7R could be bypassed by enhancing histone acetylation with the use of TSA. Bone marrow cells from IL-7Rα^−/− mice were allowed to reconstitute an irradiated fetal thymus in a hanging drop culture. The reconstituted thymus was then treated for 96 h with different concentrations of TSA. As shown in Fig. 7, TSA induced TCR-γ gene rearrangement in IL-Rα^−/− thymocytes. Fetal thymus that had not been reconstituted with bone marrow cells showed no TCR-γ rearrangement in the presence of TSA (data not shown). These data are consistent with a model that IL-7R signals control rearrangement of the TCR-γ locus via histone acetylation.

IL-7Rα^−/− mice show a severe deficiency in rearrangement of the TCR-γ locus. In this study we examine the mechanism by which IL-7Rα signals VDJ recombination of the TCR-γ locus. Based on studies in knockout mice, we observed that the γ_c component of the receptor and the γ_c-associated kinase Jak3 are required to deliver the signal from IL-7Rα to the TCR-γ locus. The production of sterile transcripts from the TCR-γ locus was greatly impaired in IL-7Rα^−/− thymocytes. This transcriptional defect could either reflect a deficiency in transcription factors or an inaccessibility of the TCR-γ locus. The latter hypothesis is favored by three observations in IL-7Rα^−/− thymocytes: (a) we readily detected the transcription factors binding to regions that have been shown to be important for expression of the TCR-γ genes; (b) the TCR-γ locus was methylated, which may obstruct its accessibility to both transcriptional and recombinational proteins; and (c) the need for IL-7Rα signals could be overcome in vitro by the use of the specific histone deacetylase inhibitor TSA.

The IL-7Rα effect on rearrangement of the TCR-γ locus is unlikely to be simply the result of the trophic effect of promoting the survival of cells undergoing the rearrangement. First, the TCR-γ locus rearranges in pro-T cells at about the same time as do the TCR-δ and -β loci. Thus, death of the pro-T cell would be expected to equally repress rearrangement of all three loci, yet the latter two loci show detectable rearrangements in IL-7Rα^−/− mice, whereas rearrangement of the TCR-γ locus is almost abrogated. Even though pro-T2 cells are virtually absent in these mice, and this is the stage in which TCR gene rearrangements normally begin, only rearrangement of the TCR-γ locus is eliminated. Second, a bcl-2 transgene was shown to promote development of the α/β lineage but not the γ/δ lineage (7, 8), so although the IL-7Rα signal also provides a trophic effect, there is no evidence that this is sufficient to sustain TCR-γ locus rearrangement. On the other hand, introducing a rearranged TCR-γ transgene only partially restored γ/δ T cell development in γ_c^−/− mice (59), reflecting the requirement for a second trophic signal from the IL-7 receptor.

The IL-7Rα chain is reported to be a component of the receptor for TSLP, a homologue of IL-7. Knockout of IL-7 (60, 61) has a less severe phenotype than knockout of IL-7Rα, in that more α/β T cells develop and γ/δ T cells are at least detectable. This has led to the suggestion that TSLP can partly compensate for the absence of IL-7 in IL-7 knockout mice, and implies that TSLP and IL-7 have similar activities. There has also been some speculation that the receptor for TSLP does not use the γ_c chain (2). Thus, if TSLP were capable of inducing T cell development, it might be independent of γ_c and Jak3, which could explain why knockout mice for γ_c and Jak3 have a less severe phenotype, more like IL-7– than IL-7Rα–deficient mice. In any case, our data show that both IL-7Rα and γ_c chains are required for the signal to rearrange the TCR-γ locus. To test whether receptors from another cytokine family could induce rearrangement of the TCR-γ locus, we have injected IL-7R^−/− mice with oncostatin M. However, we have not detected rearrangement of the locus as of the writing of this paper.

To determine whether the TCR-γ enhancer is the target of IL-7R stimulation, we examined nuclear proteins from IL-7R^−/− thymocytes. We did not find a deficiency in DNA binding based on gel mobility shift assays. However, the binding of a transcription factor to DNA does not necessarily indicate that it is transcriptionally active. Cross-linking of the IL-7R has been shown to activate the transcription factor STAT5, inducing its translocation to the nucleus (29), and it has been shown that there is a deficiency in nuclear STAT5 in IL-7R^−/− thymocytes (62). We have confirmed the latter finding that IL-7R^−/− thymocytes were deficient in nuclear proteins that bind a STAT5 motif (data not shown). STAT5 seemed a likely candidate for the relevant IL-7–induced transcription factor, since one of the enhancer sites resembles a consensus binding site for STAT5. However, we observed that the complex that actually binds this enhancer site in vivo does not contain STAT5a or -b based on a failure of specific antibodies to block binding in vitro. These findings argue against the STAT5 pathway being involved in rearrangement of the TCR-γ locus, which is also consistent with the observation of the relatively normal T cell development that occurs in STAT5a^−/−b^−/− mice (63).

It has recently been suggested that the IL-7 effect on rearrangement of the IgH locus may be via the transcription factor Pax5 (12) based on several findings: (a) Pax5 binds a site in the IgH locus; (b) Pax5^−/− mice resemble IL-7Rα^−/− mice in that the IgH locus successfully rearranges D to J, but fails to normally rearrange V to D; (c) IL-7Rα^−/− B cells showed reduced expression of Pax5. However, we detected normal levels of Pax5 transcripts in pro-T cells from IL-7Rα^−/− mice (data not shown), suggesting that its regulation is dependent on IL-7R in the B but not the T lineage, and arguing against it mediating the signal to rearrange the TCR-γ locus. That study parallels ours in noting that IL-7Rα^−/− mice were defective in producing sterile transcripts from the IgH locus and proposing that IL-7R regulated accessibility of that locus. Since thymocytes normally perform D_H to J_H but not V_H to D_H rearrangements, we tested whether TSA treatment would overcome the latter block. TSA did not induce V_H to D_H rearrangements in normal thymocytes when used under similar conditions to those in Fig. 7 (data not shown), indicating that histone deacetylation is not sufficient to account for the suppression of this rearrangement in the T cell lineage.

Our data favor the hypothesis that IL-7R signals an opening of the TCR-γ locus accompanied by locus-specific demethylation, with one site being in the enhancer, which could be a major controlling region. This idea is supported by the observations that in cell lines, methylated minichromosomes show repressed VDJ recombination (56) and that in transgenic mice, methylation of the transgene represses its rearrangement (64). A number of molecular mechanisms have been suggested to participate in transcriptional repression by DNA methylation (for review see reference 55). Methylated DNA binds proteins such as MeCP2 (which is essential for embryogenesis), which can silence chromatin over a considerable distance; for example MeCP2 separated by 1,774 bp from a transcriptional start site reduced transcription >70% (65). MeCP2 is thought to mediate this repression by attracting histone deacetylase to the region (57). Our data supports the idea that IL-7 controls the acetylation status of histones resulting in “active” chromatin. To determine how rapidly IL-7 induced demethylation of the TCR-γ locus, it would be useful to treat IL-7^−/− mice with IL-7, then monitor the methylation status of the locus. The alteration of chromatin by IL-7R signals may then allow access for transcription as well as cleavage by the Rag proteins. It remains to be determined which cis-acting elements in the TCR-γ locus respond to the IL-7 receptor signal, how they induce demethylation, whether this can cause specific histone acetylation of the TCR-γ locus, and whether this causes a direct increase of locus accessibility.

We are grateful to R. Wiles for technical assistance and to J.J. Oppenheim for comments on the manuscript.

NF

nuclear factor

TSA

trichostatin A

TSLP

thymic stromal-derived lymphopoietin