The transcription factor STAT5, which is activated by IL-7R, controls chromatin accessibility and rearrangements of the TCRγ locus. Although STAT-binding motifs are conserved in Jγ promoters and Eγ enhancers, little is known about their precise roles in rearrangements of the TCRγ locus in vivo. To address this question, we established two lines of Jγ1 promoter mutant mice: one harboring a deletion in the Jγ1 promoter, including three STAT motifs (Jγ1PΔ/Δ), and the other carrying point mutations in the three STAT motifs in that promoter (Jγ1PmS/mS). Both Jγ1PΔ/Δ and Jγ1PmS/mS mice showed impaired recruitment of STAT5 and chromatin remodeling factor BRG1 at the Jγ1 gene segment. This resulted in severe and specific reduction in germline transcription, histone H3 acetylation, and histone H4 lysine 4 methylation of the Jγ1 gene segment in adult thymus. Rearrangement and DNA cleavage of the segment were severely diminished, and Jγ1 promoter mutant mice showed profoundly decreased numbers of γδ T cells of γ1 cluster origin. Finally, compared with controls, both mutant mice showed a severe reduction in rearrangements of the Jγ1 gene segment, perturbed development of γδ T cells of γ1 cluster origin in fetal thymus, and fewer Vγ3+ dendritic epidermal T cells. Furthermore, interaction with the Jγ1 promoter and Eγ1, a TCRγ enhancer, was dependent on STAT motifs in the Jγ1 promoter. Overall, this study strongly suggests that direct binding of STAT5 to STAT motifs in the Jγ promoter is essential for local chromatin accessibility and Jγ/Eγ chromatin interaction, triggering rearrangements of the TCRγ locus.

V(D)J recombination of lymphocyte AgR genes is carried out by conserved recombinatorial signals and RAG recombinases. The accessibility model postulates that, in developing lymphocytes, specific molecular mechanisms exist to make the appropriate AgR loci accessible to the common recombination machinery in a lineage- and stage-specific manner (1). At least two kinds of cis-regulatory elements control chromatin accessibility. First, enhancer elements govern locus-wide accessibility; deletion of their respective enhancers greatly reduces rearrangements at the IgH, Igκ, TCRβ, and TCRα loci (27). Second, germline promoters control local chromatin accessibility: deletion of Dβ and Jα germline promoters severely impairs rearrangements of their respective segments (8, 9). In addition to chromatin accessibility, higher-order chromatin architecture, such as repositioning of AgR loci within the nucleoplasm and formation of chromatin loops, plays a significant role in V(D)J recombination (10).

Several transcription factors function in accessibility control through cis-regulatory elements. For example, Oct-1 recruits STAT5 to distal VH gene segments and controls their accessibility and recombination (11). IRF4 plays a role in accessibility of the IgL loci and counteracts association of an Igκ allele with heterochromatin (12). Ikaros also controls accessibility and compaction of the IgH locus (13). However, it is not known whether a given transcriptional factor has a direct or indirect effect on chromatin accessibility. In contrast, one study reported that rearrangement of the Igκ locus was decreased by targeted mutation of E-boxes in the intronic iEκ enhancer, suggesting that E2A directly interacts with the enhancer to promote recombination at the Igκ locus (14). Nonetheless, it remains unclear whether direct binding of other transcription factors to given cis-regulatory elements is essential for the accessibility control of AgR loci in vivo.

IL-7R controls accessibility of the TCRγ locus. V-J recombination and germline transcription of the TCRγ locus are severely impaired in mice deficient in the gene that encodes IL-7Rα (1517). In vitro and ex vivo experiments indicate that, once activated by IL-7R, STAT5 binds to its consensus motifs in Jγ germline promoters (17). STAT5 then recruits transcriptional coactivators and induces histone acetylation, germline transcription, and recombination of Jγ gene segments (18). STAT5 also binds to conserved STAT motifs in TCRγ 3′ enhancers (Eγ) and a DNase I hypersensitivity site (HsA) in this locus (19), augmenting enhancer activity (20, 21). Nevertheless, it is not known whether direct binding of STAT5 to conserved STAT motifs in Jγ promoters plays a causative role in chromatin accessibility and recombination of the TCRγ locus in vivo. TCRγ rearrangements and γδ T cell development are completely blocked in IL-7Rα– or STAT5-deficient mice (15, 22, 23), making it technically difficult to address these issues.

To elucidate the role of STAT5 recruitment to the Jγ germline promoters, we established and analyzed two lines of Jγ1 promoter mutant mice: one harbored a deletion in the Jγ1 promoter, including three STAT motifs, and the other carried point mutations in the three STAT motifs found in that promoter. Both types of mutations severely reduced chromatin accessibility and rearrangement of the Jγ1 gene segment, as well as perturbed development of γδ T cells of γ1 cluster origin. In addition, interaction of Eγ1 and Jγ1 was impaired in STAT motif mutant mice. Thus, to our knowledge, this study is the first report of Jγ mutant mice, demonstrating absolute dependence of V-J recombination on Jγ1 promoter and STAT5 recruitment. Our findings strongly suggest that direct binding of STAT5 to STAT motifs in Jγ promoters triggers local chromatin accessibility and rearrangements of Jγ gene segments.

C57BL/6 mice were purchased from Japan SLC. RAG2-deficient mice on a C57BL/6 background were obtained from Dr. M. Ito at Central Laboratories for Experimental Animals, Kawasaki, Japan (a kind gift of Dr. F.W. Alt, Harvard Medical School, Boston, MA). Mice were maintained under specific pathogen–free conditions in the Experimental Research Center for Infectious Diseases at the Institute for Virus Research, Kyoto University. All mouse protocols were approved by Kyoto University.

Targeting vectors were constructed using Red recombination technology with a murine bacterial artificial chromosome (BAC) clone containing the TCRγ1 cluster (RP23-167F18), as described (24). One loxP sequence was inserted 171 bp upstream of the Jγ1 gene segment (46 bp downstream of the major transcription start site of the Jγ1 germline promoter), and the neomycin resistance gene cassette flanked by FRT sequences on both sides and one loxP sequence at the 3′ end was inserted 1108 bp upstream of the Jγ1 gene segment, with three wild-type (TTCNNNGAA) or mutated (TTCNNNTCC) STAT motifs in the Jγ1 germline promoter (Supplemental Fig. 1A, 1B). The targeting vector was retrieved from a modified BAC clone with the 2.1-kb 5′-homologous fragment, the neomycin resistance gene cassette, and a 5.4-kb 3′-homologous fragment, flanked by diphtheria toxin A subunit cDNA. Linearized targeting vectors were introduced into the KY1.1 embryonic stem (ES) cell line derived from C57BL/6 × 129S6/SvEvTac mouse embryos (a gift of Dr. Junji Takeda, Osaka University) (25). Homologous recombinants were screened by PCR and confirmed by Southern blot analysis with 5′ and 3′ probes (Supplemental Fig. 1C, 1D). The neomycin resistance gene cassette and the Jγ1 germline promoter were removed from the recombinant allele by infecting targeted ES clones in vitro with the adenovirus expressing Flp and Cre recombinases, AxCAFLP and AdV-Cre, respectively (gifts of Dr. Izumu Saito, Institute of Medical Science, University of Tokyo) (26, 27), and targeted clones were injected into ICR eight-cell embryos. Chimeric mice were backcrossed onto C57BL/6 mice at least four times. The following primers were used for Jγ1PΔ/Δ mouse genotyping: Jγ1/F4, 5′-CAGTCGACTCAGACAGACATGAGGAGGT-3′ and Jγ1/R2, 5′-CAGCGGCCGCTAAATACACAACTTCTATGTTTTCT-3′.

CD3CD4CD8 triple-negative (TN) thymocytes were isolated as previously described (18). Epidermal cells were isolated from the ears of mice as described, with slight modifications (28). Briefly, ears were separated into two faces with fine forceps and incubated in PBS containing 0.05% trypsin and 0.5 mM EDTA for 60 min at 37°C. Epidermal sheets were separated from the dermis with fine forceps, and epidermal cells were released by vortexing in PBS. Viable cells were purified by density gradient centrifugation with Lympholyte-M (CEDARLANE Laboratories) and cultured overnight in RPMI 1640 medium containing 10% FBS and 20 U/ml recombinant human IL-2 (Shionogi & Co.). For IL-7 stimulation, 10% v/v IL-7 culture supernatant of J558–IL-7 cells was used (a kind gift of Dr. H. Karasuyama, Tokyo Medical and Dental University, Tokyo, Japan).

To quantify V-J coding joints, genomic DNA was digested with BamHI to reduce viscosity and then purified. To quantify germline TCRγ transcripts, total RNA was reverse transcribed using random primers. Genomic DNA or cDNA was amplified using an ABI 7500 Sequence Detector (Applied Biosystems) with a QuantiTect SYBR Green PCR Kit (QIAGEN) with ROX reference dye (Invitrogen). PCR was carried out at 95°C for 15 min, followed by 40 cycles consisting of 95°C for 20 s, 55°C for 30 s, and 72°C for 1 min. After amplification, melting curve analysis was performed to verify reaction specificity. In each experiment, samples were analyzed in duplicate or triplicate. Primers for coding joints were Vγ1.1/F1, 5′-GACAGATGAGAGTGCGCAAA-3′; Jγ4/R1, 5′-TCTTGACCCATGATGTGCCT-3′; Vγ1.2/F1, 5′-CCGGCAAAAAACAAATCAAC-3′; Jγ2/R1, 5′-GGGAATTACTATGAGCTTT-3′; Vγ2-probe 1 (Vγ2-gF) (29); Jγ1 (30); Vγ3 (30); Vγ4-probe 1, 5′-GTCTTCAGTCCTCACCATAC-3′; V5-1 (31); Jγ1-1 (18); IL-7RII-5′, 5′-GGAGACCTAGAAGATGCAGACG-3′; and IL-7RII-3′, 5′-AGAGAGAGACAGGAGATGGATTC-3′. Primers for germline transcripts and chromatin immunoprecipitation (ChIP) were 5′Jγ1/5′-3, 5′-ACAATTTATATGCAATGACT-3′; Jγ1-1 (18); 5′Jγ2/F1, 5′-CTCAGGACATAAATGTCTGG-3′; Jγ2/R1, 5′-GGGAATTACTATGAGCTTT-3′; Jγ4/F2 (Jγ4-F) (31); Jγ4/R2 (Jγ4-R) (31); Vγ1.1/Fw1, 5′-AGAGAGACAGATGAGAGTGC-3′; Vγ1.1/Rw1, 5′-GCAATGAAGACTCAGGTGGG-3′; Vγ1.2/F1, 5′-CCGGCAAAAAACAAATCAAC-3′; Vγ1.2/R2, 5′-CTGTGAGCAATGAAGGCCCT-3′; Vγ2-probe 1 (Vγ2-gF) (29); Vγ2-3′b (Vγ2-gR) (29); Vγ3/RT-sense (31); V3S/R3 (31); V4S-F3 (31); V4-3′a (31); V5S/F1 (31); V5S/R3 (Vγ5-R) (31); HsA2/F1 (31); HsA2/R3 (31); Eγ1/F1 (29); Eγ1/3′-1 (29); 5′Jκ1/5′-4 (32); 3′Jκ1/3′-1 (32); mHPRT-F1 (33); and mHPRT-R1 (33).

Ligation-mediated (LM)-PCR was performed as previously described (18, 34). Genomic DNA from TN thymocytes was ligated to the BW linker. Five-fold serial dilutions of ligated DNA were amplified for 12 cycles with the distal 11 locus-specific primer and the linker-specific BW-1H primer. The first PCR product was amplified for 30 cycles (Jγ1) or 35 cycles (Dβ1 and Jβ1) with proximal locus-specific primers and the BW primer. For Vγ2 or Vγ1.1 cleavages, DNA ligated with BW linker was measured by real-time PCR and normalized with the amounts of input genomic DNA (IL-7R). The following primers were used for LM-PCR: Jγ1: first PCR, J1P-F1 (31) and BW-1H (18) and second PCR, J1P/F3 (31) and BW-1H3 (18); Dβ1: first PCR, 3′Dβ1 3′-1 (18) and BW-1H and second PCR, 3′Dβ1 3′-2 (18) and BW-1H3; Jβ1: first PCR, 5′Jβ 5′-3 (18) and BW-1H and second PCR, 5′Jβ1 5′-4 (18) and BW-1H4 (18); Vγ2: V2S-R5, 5′-CGTCGTTGCATGGAAAGTTC-3′, and BW-1H; and Vγ1.1: Vγ1.1-LM2, 5′-AGTTGGTAACCTGTAGGACC-3′, and BW-1H.

ChIP was performed as described previously (18). Briefly, splenocytes and thymocytes of Jγ1Pf/f, Jγ1PΔ/Δ, and Jγ1PmS/mS mice on a RAG2−/− background were fixed under the following conditions: for acetylated or methylated histone H3, 0.7% formaldehyde for 5 min at room temperature; for STAT5, 1% formaldehyde for 8 min at room temperature and 20 min at 4°C; and for BRG1, 1% formaldehyde for 10 min at room temperature. Soluble chromatin was immunoprecipitated with 3 μg anti-acetylated H3 Ab (Millipore); 1.5 μl each anti-STAT5A Ab and anti-STAT5B Ab (R&D Systems); 50 μl anti-monomethylated H3K4 Ab (CMA302), anti-dimethylated H3K4 Ab (CMA303), or anti-trimethylated H3K4 Ab (CMA304) (35); 5 μl anti-SNF2β/BRG1 antiserum (Millipore); 2–3 μg normal rabbit IgG (MBL); 2 μg normal mouse IgG (Jackson ImmunoResearch Laboratories); or 5 μl normal rabbit serum (Sigma-Aldrich) overnight. Purified ChIP and input DNAs were measured by real-time PCR, as described above.

The following fluorescent dye– or biotin-conjugated Abs were used: CD3 (145-2C11), TCRβ (H57-597), TCRγδ (GL-3), I-A/I-E (M5/114.15.2), and streptavidin-PE (purchased from BioLegend or eBioscience). The following Abs were purified from hybridoma supernatants and labeled with fluorescent dye or biotin: Vγ1.1 (2.11), Vγ2 (UC3), Vγ3 (M181.8), and Vγ5 (GL1). Stained cells were analyzed with a FACSCalibur flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (TreeStar). In all figures, values in quadrants and gated areas indicate percentages in each population.

Immunofluorescence staining of epidermal sheets was done as described previously (28). Briefly, sheets were fixed in cold acetone for 10 min, rinsed in PBS, and incubated with PE–anti-γδTCR (GL-3) or Alexa Fluor 555–anti-Vγ3 (M181.1) and FITC–anti-I-A/I-E (M5/114.15.2) Abs for 2 h at room temperature. After rinsing in PBS, sheets were mounted on slide glass and examined under a confocal laser scanning microscope (Leica TCS SP5 or Leica TCS SP8; Leica Microsystems). Dendritic epidermal T cells (DETCs) and Langerhans cells were counted, and their density was reported as the mean cell number/mm2 of three areas.

Thymocytes and splenocytes from Jγ1Pf/f RAG2−/− and Jγ1PmS/mS RAG2−/− mice were stimulated with IL-7 for 30 min at 37°C and then subjected to a chromosome conformation capture (3C) assay, as previously described (36, 37). Formaldehyde–cross-linked chromatin was digested with DpnII (New England Biolabs). Digested samples with >70% digestion efficiency were followed by intramolecular ligation with T4 DNA ligase (Takara Bio). Samples were reverse cross-linked, purified with a Wizard DNA Clean-Up System (Promega), and quantified using an ABI StepOnePlus (Applied Biosystems) with a QuantiTect SYBR Green PCR Kit. To prepare control templates for quantitative PCR, we modified the Jγ1Pf+Neo targeting vector (Supplemental Fig. 1A), because insertion of the 3′ loxP site created an artificial DpnII site in Jγ1Pf and Jγ1PmS mice. The neomycin resistance gene cassette in the Jγ1Pf+Neo BAC was excised in Escherichia coli strain EL250 that expresses Flp recombinase by arabinose treatment. Neomycin resistance cassette–excised vector (Jγ1Pf) was digested with Sau3AI, which is an isoschizomer of DpnII but insensitive to Dam methylation, followed by random ligation. The following primers were used for the 3C assay: anchor primer (Jγ1p/3C DpnII/R), 5′-CTTTATTGAGAGCAACCCGG-3′; primer 1 (Jγ1p up/3C DpnII/R2), 5′-GCAGCTTAAGATTTCAACCC-3′; primer 2 (Jγ1-Cγ1/3C DpnII/R), 5′-TAGAGCTTTCCCTGTCCCAG-3′; primer 3 (Cγ1/3C DpnII/R), 5′-ATCCCTTCACAACCCTACC-3′; primer 4 (Cγ1-Eγ1/3C DpnII/R), 5′-CCTCTAATCTGTGAACTGTGG-3′; and primer 5 (Eγ1/3C DpnII/R), 5′-ATCAAGACTCCTGCCTATTG-3′.

An unpaired two-tailed Student t test was used for all statistical analyses.

To investigate the roles of STAT consensus motifs found in Jγ germline promoters in accessibility control of the TCRγ locus in vivo, we focused on the most characterized γ1 cluster (Fig. 1A). We established two kinds of Jγ1 promoter mutant mice, as well as Jγ1 promoter-floxed control mice (Jγ1Pf/f). One mutant line (Jγ1PΔ/Δ) had a deletion in a 940-bp fragment of the Jγ1 promoter, including three STAT motifs; the other (Jγ1PmS/mS) carried point mutations within those three STAT motifs (Supplemental Fig. 1A, 1B). Homologous recombination was confirmed by Southern blot analysis (Supplemental Fig. 1C, 1D), and deletions of the neomycin cassette and the promoter were verified by PCR (Supplemental Fig. 1E).

FIGURE 1.

STAT5 recruitment and germline transcription are severely reduced at the Jγ1 gene segment in Jγ1 promoter mutant mice. (A) Schematic illustration of murine TCRγ locus. Exons and pseudogenes are depicted as solid and open boxes, respectively. Eγ and HsA are depicted as open ovals. STAT consensus motifs (●) are conserved in the Jγ germline promoters, Eγ, and HsA elements. (B) Jγ1Pf/f splenocytes and thymocytes and Jγ1PmS/mS thymocytes on a RAG2−/− background were cultured with (+) or without (−) IL-7 stimulation, and soluble chromatin preparations were immunoprecipitated with anti-STAT5 Ab or control normal rabbit IgG. Purified ChIP and input DNAs were analyzed by real-time PCR. The amount of ChIP DNA was normalized to that of input DNA. Values are the mean ± SEM of duplicate data points. Data represent two independent experiments with similar results. (C) Germline transcripts from the TCRγ locus were measured by real-time RT-PCR in Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f thymocytes on a RAG2−/− background. Transcript levels were normalized to HPRT mRNA, and transcript levels relative to Jγ1Pf/f RAG2−/− mice are shown as the mean ± SEM of four (Vγ) or five (Jγ) experiments. ****p < 0.001. n.s., not significant.

FIGURE 1.

STAT5 recruitment and germline transcription are severely reduced at the Jγ1 gene segment in Jγ1 promoter mutant mice. (A) Schematic illustration of murine TCRγ locus. Exons and pseudogenes are depicted as solid and open boxes, respectively. Eγ and HsA are depicted as open ovals. STAT consensus motifs (●) are conserved in the Jγ germline promoters, Eγ, and HsA elements. (B) Jγ1Pf/f splenocytes and thymocytes and Jγ1PmS/mS thymocytes on a RAG2−/− background were cultured with (+) or without (−) IL-7 stimulation, and soluble chromatin preparations were immunoprecipitated with anti-STAT5 Ab or control normal rabbit IgG. Purified ChIP and input DNAs were analyzed by real-time PCR. The amount of ChIP DNA was normalized to that of input DNA. Values are the mean ± SEM of duplicate data points. Data represent two independent experiments with similar results. (C) Germline transcripts from the TCRγ locus were measured by real-time RT-PCR in Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f thymocytes on a RAG2−/− background. Transcript levels were normalized to HPRT mRNA, and transcript levels relative to Jγ1Pf/f RAG2−/− mice are shown as the mean ± SEM of four (Vγ) or five (Jγ) experiments. ****p < 0.001. n.s., not significant.

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First, we confirmed by ChIP analysis whether mutations in the STAT motifs inhibited STAT5 recruitment to the Jγ1 promoter. We observed that STAT5 recruitment to the Jγ1 promoter was higher in Jγ1Pf/f RAG2−/− control thymocytes than in Jγ1PmS/mS RAG2−/− thymocytes (Fig. 1B). Although the association of STAT5 was greatly increased after IL-7 stimulation in Jγ1Pf/f RAG2−/− control thymocytes, mutations in STAT motifs completely blocked STAT5 binding to the Jγ1 promoter in Jγ1PmS/mS RAG2−/− thymocytes. These results indicate that the mutations in the STAT motifs abrogate STAT5 binding to the Jγ1 promoter in Jγ1 promoter mutant mice.

We then used real-time PCR to determine whether introduction of loxP and FRT sequences into the Jγ1 germline promoter had any influence. Although the recruitment of STAT5 to Jγ1 promoter and histone acetylation in Jγ1Pf/f × RAG2−/− mice was comparable to that in RAG2−/− mice (Supplemental Fig. 2A, 2B), germline transcription of Jγ1 was reduced by 60% (Supplemental Fig. 2C). Consistent with this, levels of Vγ5-Jγ1 and Vγ2-Jγ1 signal joints were modestly reduced in Jγ1Pf/f thymocytes compared with wild-type thymocytes (Supplemental Fig. 2D). DNA cleavage at Jγ1 and Vγ2 was slightly reduced, likely due to coupled cleavage of these segments, whereas that of Vγ1.1 was unchanged in Jγ1Pf/f mice (Supplemental Fig. 2E, 2F). Nonetheless, we observed no significant difference in the number or development of γδ T cells, with the exception that the number of Vγ1.1+ T cells was slightly increased (Supplemental Fig. 2G). Thus, we conclude that insertion of loxP and FRT sequences had modest effects on rearrangements of the Jγ1 gene segment in Jγ1Pf/f mice.

To assess accessibility of the TCRγ locus, we first analyzed germline transcription in Jγ1 promoter mutant mice on a RAG2−/− background. Levels of germline transcripts from the Jγ1 gene segment were severely reduced in both Jγ1PΔ/Δ and Jγ1PmS/mS mice compared with Jγ1Pf/f mice (>600- and >2700-fold, respectively) (Fig. 1C). In contrast, we observed no significant difference in germline transcripts from other Jγ and Vγ gene segments. These results suggest that direct binding of STAT5 to the Jγ1 promoter plays an essential role in germline transcription of that segment.

Next, we used ChIP assay to assess the histone H3 acetylation (H3ac) status of the TCRγ locus in both types of Jγ1 promoter mutant mice on a RAG2−/− background. We detected substantial levels of H3ac in Jγ gene segments in Jγ1Pf/f mice (Fig. 2A). In contrast, H3ac was severely reduced in the Jγ1 gene segment in both Jγ1PΔ/Δ and Jγ1PmS/mS mice relative to Jγ1Pf/f control mice. As in the case of germline transcription, levels of H3ac were unchanged or slightly reduced in other Jγ and Vγ gene segments of Jγ1PΔ/Δ and Jγ1PmS/mS mice.

FIGURE 2.

H3ac and H3K4 methylation of the Jγ1 gene segment is severely reduced in Jγ1 promoter mutant mice. Soluble chromatin preparations of Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f thymocytes on a RAG2−/− background were immunoprecipitated with anti-AcH3 Ab (A), anti-H3K4me3 Ab (B), or control IgG. Soluble chromatin preparations of Jγ1Pf/f splenocytes and thymocytes and Jγ1PmS/mS thymocytes on a RAG2−/− background were immunoprecipitated with anti-H3K4me2 Ab (C), anti-H3K4me1 Ab (D), or control IgG. Purified ChIP and input DNAs were analyzed by real-time PCR. The amount of ChIP DNA was normalized to that of input DNA. Values are the mean ± SEM of duplicate data points. Data represent three (A) or two (B–D) independent experiments with similar results. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. n.s., not significant.

FIGURE 2.

H3ac and H3K4 methylation of the Jγ1 gene segment is severely reduced in Jγ1 promoter mutant mice. Soluble chromatin preparations of Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f thymocytes on a RAG2−/− background were immunoprecipitated with anti-AcH3 Ab (A), anti-H3K4me3 Ab (B), or control IgG. Soluble chromatin preparations of Jγ1Pf/f splenocytes and thymocytes and Jγ1PmS/mS thymocytes on a RAG2−/− background were immunoprecipitated with anti-H3K4me2 Ab (C), anti-H3K4me1 Ab (D), or control IgG. Purified ChIP and input DNAs were analyzed by real-time PCR. The amount of ChIP DNA was normalized to that of input DNA. Values are the mean ± SEM of duplicate data points. Data represent three (A) or two (B–D) independent experiments with similar results. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. n.s., not significant.

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Histone H3 lysine 4 (H3K4) trimethylation (H3K4me3) is known to correlate with transcriptional activity. In addition, RAG2 recombinase binds to H3K4me3-modified nucleosome, and that binding is essential for DNA cleavage occurring in V(D)J recombination (3840). Thus, we analyzed H3K4me3 status in Jγ1 promoter mutant mice. Levels of H3K4me3 were specifically and significantly diminished in the Jγ1 gene segment of Jγ1PΔ/Δ and Jγ1PmS/mS mice relative to Jγ1Pf/f control mice (Fig. 2B).

H3K4 dimethylation (H3K4me2) often correlates with active promoters and is premarked before H3K4me3, H3ac, and histone acetyltransferase recruitment (41, 42). Thus, we then analyzed H3K4me2 in Jγ1 promoter mutant mice. The levels of H3K4me2 were higher in thymocytes than in splenocytes of Jγ1Pf/f RAG2−/− control mice in the entire TCRγ locus, with the exception of the Vγ5 gene (Fig. 2C). However, in Jγ1PmS/mS RAG2−/− thymocytes, the levels of H3K4me2 at the Jγ1 gene segment were specifically reduced to the levels of Jγ1Pf/f RAG2−/− splenocytes.

Because it is suggested that H3K4 monomethylation (H3K4me1) is marked prior to H3K4me2 and H3K4me3 in lineage-determining genes before their expression (43, 44), we next analyzed H3K4me1. Similarly to the other histone modifications, the levels of H3K4me1 at the Jγ1 gene segment were significantly reduced in Jγ1PmS/mS RAG2−/− thymocytes compared with Jγ1Pf/f RAG2−/− thymocytes (Fig. 2D). Unlike H3ac and H3K4me2 (32), the levels of H3K4me1 were high at fetal-type Vγ4 and Vγ3 gene segments in wild-type adult thymocytes, indicating that H3K4me1 alone does not necessarily correlate with chromatin accessibility. Taken together, these results demonstrate that Jγ1PmS/mS mice show a specific reduction in germline transcription and active histone modifications at the Jγ1 gene segment and suggest that STAT5 binding to the Jγ1 promoter triggers a cascade of local epigenetic changes by targeting the earliest active histone modification, H3K4me1.

The SWI/SNF chromatin-remodeling complex is recruited to the TCRβ and IgH loci and is essential for chromatin accessibility and V(D)J recombination (45, 46). To test whether recruitment of the SWI/SNF complex is attenuated in Jγ1 promoter mutant mice, we analyzed the association of BRG1, a catalytic subunit of the SWI/SNF complex, with the TCRγ locus by ChIP assay. Levels of BRG1 binding were high in the entire TCRγ locus in thymocytes, but not in splenocytes, of Jγ1Pf/f RAG2−/− mice (Fig. 3A). Mutations in STAT motifs of the Jγ1 promoter specifically reduced the association of BRG1 with the Jγ1 gene segment. These results suggest that STAT5 binding to the STAT motifs is essential for recruitment of the SWI/SNF complex to the Jγ1 promoter. Unexpectedly, BRG1 was recruited to the Vγ5 gene segment in splenocytes, as well as in thymocytes, of Jγ1Pf/f RAG2−/− mice. A similar chromatin status at the Vγ5 gene segment also was observed for H3K4me2 in RAG2−/− splenocytes (Fig. 2C).

FIGURE 3.

Association of SWI/SNF complexes and Eγ1 with Jγ1 gene segment is substantially reduced in Jγ1 promoter mutant mice. (A) Soluble chromatin preparations of Jγ1Pf/f splenocytes and thymocytes and Jγ1PmS/mS thymocytes on a RAG2−/− background were immunoprecipitated with anti-BRG1 Ab or control normal rabbit serum. Purified ChIP and input DNAs were analyzed by real-time PCR. The amount of ChIP DNA was normalized to that of input DNA. Values are the mean ± SEM of duplicate data points. Data represent two independent experiments with similar results. (B) Schematic illustration between Jγ1 upstream and Eγ1 (top) and 3C data (bottom). Thymocytes and splenocytes from Jγ1Pf/f and Jγ1PmS/mS mice on a RAG2−/− background were stimulated with IL-7 at 37°C for 30 min and subjected to a 3C assay. Interaction between Jγ1 promoter (anchor primer) and indicated fragments (gray bars, primers 1–5) was quantified. The quantity of DNA was normalized to the DpnII-insensitive region. Boxes with dotted lines indicate artificially inserted sequences (mean ± SEM, n = 10). (C) Jγ1 germline transcripts were measured in Eγ1+/+ and Eγ1−/− thymocytes on a RAG2−/− background by real-time RT-PCR. Transcript levels were normalized to HPRT mRNA. The transcript levels relative to Eγ1+/+ RAG2−/− mice are shown as the mean ± SEM of seven experiments. ****p < 0.001. n.s., not significant.

FIGURE 3.

Association of SWI/SNF complexes and Eγ1 with Jγ1 gene segment is substantially reduced in Jγ1 promoter mutant mice. (A) Soluble chromatin preparations of Jγ1Pf/f splenocytes and thymocytes and Jγ1PmS/mS thymocytes on a RAG2−/− background were immunoprecipitated with anti-BRG1 Ab or control normal rabbit serum. Purified ChIP and input DNAs were analyzed by real-time PCR. The amount of ChIP DNA was normalized to that of input DNA. Values are the mean ± SEM of duplicate data points. Data represent two independent experiments with similar results. (B) Schematic illustration between Jγ1 upstream and Eγ1 (top) and 3C data (bottom). Thymocytes and splenocytes from Jγ1Pf/f and Jγ1PmS/mS mice on a RAG2−/− background were stimulated with IL-7 at 37°C for 30 min and subjected to a 3C assay. Interaction between Jγ1 promoter (anchor primer) and indicated fragments (gray bars, primers 1–5) was quantified. The quantity of DNA was normalized to the DpnII-insensitive region. Boxes with dotted lines indicate artificially inserted sequences (mean ± SEM, n = 10). (C) Jγ1 germline transcripts were measured in Eγ1+/+ and Eγ1−/− thymocytes on a RAG2−/− background by real-time RT-PCR. Transcript levels were normalized to HPRT mRNA. The transcript levels relative to Eγ1+/+ RAG2−/− mice are shown as the mean ± SEM of seven experiments. ****p < 0.001. n.s., not significant.

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In the TCRβ locus, Eβ interacts with the germline promoter of Dβ genes, and this interaction is dependent on two Runx-binding motifs in Eβ (37, 47). Because STAT5 is recruited to Eγ1 and increases the transcriptional activity of the Jγ1 promoter (20), we speculated that Eγ1 associates with the Jγ1 promoter via STAT5. To test this hypothesis, we used a 3C assay in Jγ1 promoter mutant mice on a RAG2−/− background. Relative cross-linking between the Jγ1 promoter and Eγ1 was higher in thymocytes of Jγ1Pf/f × RAG2−/− mice compared with thymocytes of Jγ1PmS/mS × RAG2−/− mice and splenocytes from these mice (Fig. 3B). However, germline transcription of the Jγ1 promoter was unchanged in Eγ1-deficient mice (Fig. 3C, Supplemental Fig. 3), consistent with the previous report that rearrangement of TCRγ genes was almost normal in Eγ1-deficient mice (48). These data suggest that STAT motifs in the Jγ1 promoter are essential for the interaction of Jγ1 and Eγ1, although loss of Eγ1 function might be compensated for by other cis-regulatory elements in Eγ1-deficient mice.

To assess whether impaired chromatin accessibility affects rearrangements of the Jγ1 gene segment, we analyzed Vγ-Jγ coding joints in adult thymocytes of Jγ1 promoter mutant mice. Levels of Vγ5-Jγ1 and Vγ2-Jγ1 coding joints were drastically decreased in Jγ1PΔ/Δ or Jγ1PmS/mS mice compared with Jγ1Pf/f mice (Fig. 4A). In contrast, levels of Vγ1.2-Jγ2 and Vγ1.1-Jγ4 coding joints were unchanged or slightly increased in Jγ1PΔ/Δ and Jγ1PmS/mS mice relative to controls. Because approximately two thirds of Jγ1 and Jγ2 alleles are rearranged in total adult thymocytes (15), and the majority of thymocytes are in the αβ T cell lineage, the Vγ-Jγ coding joints observed in this study likely reflect Vγ-Jγ rearrangements occurring primarily in αβ T cells.

FIGURE 4.

Rearrangements of the Jγ1 gene segment are severely impaired in Jγ1 promoter mutant mice. Vγ-Jγ coding joints were measured by real-time PCR in whole thymocytes (A) and TN thymocytes (B) of Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f mice. Coding joint levels were normalized to the amount of genomic DNA (IL-7R), and rearrangement levels relative to Jγ1Pf/f mice are shown as the mean ± SEM of two experiments. (C) Double-strand breaks were analyzed by LM-PCR. Genomic DNA from TN thymocytes of Jγ1Pf/f and Jγ1PmS/mS mice was ligated to linkers, serially diluted 5-fold, and amplified by PCR to detect broken signal ends of the Jγ1, Dβ1, and Jβ1 gene segments. Water [DNA(−)] alone or unligated [Lig(−)] genomic DNA served as negative controls. Data represent two independent experiments with similar results. (D) Double-strand breaks at Vγ2 and Vγ1.1 gene segments were analyzed by LM real-time PCR. DNA cleavage levels relative to Eγ1+/+ RAG2−/− mice are shown as the mean ± SEM (n = 2). *p < 0.05, ****p < 0.001. n.s., not significant.

FIGURE 4.

Rearrangements of the Jγ1 gene segment are severely impaired in Jγ1 promoter mutant mice. Vγ-Jγ coding joints were measured by real-time PCR in whole thymocytes (A) and TN thymocytes (B) of Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f mice. Coding joint levels were normalized to the amount of genomic DNA (IL-7R), and rearrangement levels relative to Jγ1Pf/f mice are shown as the mean ± SEM of two experiments. (C) Double-strand breaks were analyzed by LM-PCR. Genomic DNA from TN thymocytes of Jγ1Pf/f and Jγ1PmS/mS mice was ligated to linkers, serially diluted 5-fold, and amplified by PCR to detect broken signal ends of the Jγ1, Dβ1, and Jβ1 gene segments. Water [DNA(−)] alone or unligated [Lig(−)] genomic DNA served as negative controls. Data represent two independent experiments with similar results. (D) Double-strand breaks at Vγ2 and Vγ1.1 gene segments were analyzed by LM real-time PCR. DNA cleavage levels relative to Eγ1+/+ RAG2−/− mice are shown as the mean ± SEM (n = 2). *p < 0.05, ****p < 0.001. n.s., not significant.

Close modal

To further analyze rearrangements in early thymocytes, we measured levels of Vγ-Jγ coding joints in TN immature thymocytes. As seen in total thymocytes, Vγ5-Jγ1 and Vγ2-Jγ1 coding joints were dramatically decreased in TN thymocytes of Jγ1PΔ/Δ and Jγ1PmS/mS mice relative to control mice (Fig. 4B). In contrast, Vγ1.2-Jγ2 and Vγ1.1-Jγ4 coding joints were not significantly affected. These results indicate that direct binding of STAT5 to the Jγ1 promoter is essential for rearrangements of the Jγ1 gene segment.

We next determined whether the reduction in Jγ1 gene segment rearrangements is due to a block in DNA cleavage by RAG recombinases in promoter mutant mice. To do so, we used LM-PCR to assess broken signal ends in TN thymocytes from Jγ1Pf/f and Jγ1PmS/mS mice. Levels of broken signal ends of the Jγ1 gene segment were significantly reduced in Jγ1PmS/mS compared with Jγ1Pf/f mice (Fig. 4C), while levels of broken signal ends at Dβ1 and Jβ1 gene segments were unchanged. In addition to Jγ1, cleavage at Vγ2 gene segment was significantly reduced, whereas cleavage at Vγ1.1 was unchanged (Fig. 4D). These results demonstrate that DNA cleavage at the Jγ1 gene segment and corresponding Vγ gene segment is specifically impaired in Jγ1PmS/mS mice, suggesting that the direct binding of STAT5 to the Jγ1 promoter is essential for chromatin accessibility of the Jγ1 gene segment to the RAG machinery and for coupled cleavage at corresponding Vγ gene segments.

Next, we analyzed the development of γδ T cells in Jγ1 promoter mutant mice by flow cytometry. Although αβ T cells in the thymus of Jγ1PΔ/Δ and Jγ1PmS/mS mice were not affected, the number of γδ T cells was slightly decreased relative to controls (Fig. 5A, 5B). Particularly, Vγ2+ T cells were severely reduced in both mutant mouse genotypes, whereas Vγ1.1+ T cells were slightly increased. In the periphery, the proportion and number of γδ T cells among intraepithelial lymphocytes (IELs) of the small intestine tended to be decreased in Jγ1PΔ/Δ and Jγ1PmS/mS mice, although not significantly (Fig. 5C, 5D). Only a small number of Vγ2+ and Vγ5+ T cells was detected in IELs of Jγ1PΔ/Δ and Jγ1PmS/mS mice, whereas Vγ1.1+ T cells were unchanged. A severe reduction in the number of Vγ2+, but not Vγ1.1+, T cells also was observed in the spleen and lymph nodes of Jγ1PΔ/Δ and Jγ1PmS/mS mice relative to controls (Supplemental Fig. 4A). These results demonstrate that development of γδ T cells of γ1 cluster origin is severely and specifically impaired in Jγ1 promoter mutant mice and is consistent with reductions in rearrangements of the Jγ1 gene segment.

FIGURE 5.

Development of γδ T cells of γ1 cluster origin is severely impaired in Jγ1 promoter mutant mice. (A) Thymocytes of Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f mice were stained with anti-CD3, anti-TCRβ, and anti-TCRγδ Abs or with anti-CD3 and anti-Vγ2 or anti-Vγ1.1 Abs. (B) Absolute numbers of T cell subpopulations (mean ± SEM, n = 5–8, at 4–5 wk old). (C) IELs of Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f mice were stained with anti-CD3, anti-TCRβ, and anti-TCRγδ Abs or with anti-CD3 and anti-Vγ5, anti-Vγ2, or anti-Vγ1.1 Abs. (D) Absolute numbers of IEL subpopulations (mean ± SEM, n = 5–7, at 4–5 wk old). *p < 0.05, ***p < 0.005, ****p < 0.001. n.s., not significant.

FIGURE 5.

Development of γδ T cells of γ1 cluster origin is severely impaired in Jγ1 promoter mutant mice. (A) Thymocytes of Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f mice were stained with anti-CD3, anti-TCRβ, and anti-TCRγδ Abs or with anti-CD3 and anti-Vγ2 or anti-Vγ1.1 Abs. (B) Absolute numbers of T cell subpopulations (mean ± SEM, n = 5–8, at 4–5 wk old). (C) IELs of Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f mice were stained with anti-CD3, anti-TCRβ, and anti-TCRγδ Abs or with anti-CD3 and anti-Vγ5, anti-Vγ2, or anti-Vγ1.1 Abs. (D) Absolute numbers of IEL subpopulations (mean ± SEM, n = 5–7, at 4–5 wk old). *p < 0.05, ***p < 0.005, ****p < 0.001. n.s., not significant.

Close modal

Rearrangement within the γ1 cluster is differentially controlled during development, and Vγ3 and Vγ4 gene segments are preferentially rearranged in fetal thymus (49). Thus, we analyzed Vγ-Jγ coding joints in fetal thymocytes of Jγ1 promoter mutant mice. Levels of Vγ3-Jγ1 and Vγ4-Jγ1, as well as Vγ2-Jγ1 and Vγ5-Jγ1, coding joints were severely reduced in embryonic day 17.5 fetal thymus of Jγ1PΔ/Δ and Jγ1PmS/mS mice compared with Jγ1Pf/f control mice (Fig. 6A). In contrast, Vγ1.2-Jγ2 and Vγ1.1-Jγ4 coding joints were slightly increased in both types of mutant mice. These results demonstrate that direct binding of STAT5 to the Jγ1 promoter is essential for rearrangement of the Jγ1 gene segment at the fetal stage.

FIGURE 6.

Rearrangements of the Jγ1 gene segment and development of γδ T cells of γ1 cluster origin are severely impaired in fetal thymus of Jγ1 promoter mutant mice. (A) Vγ-Jγ coding joints were measured by real-time PCR in Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f embryonic day 17.5 fetal thymocytes. Coding joint levels were normalized to those of genomic DNA (IL-7R), and rearrangement levels relative to that of Jγ1Pf/f mice are shown as the mean ± SEM of two to four experiments. (B) Embryonic day 17.5 thymocytes of Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f mice were stained with anti-CD3 and anti-TCRγδ, anti-Vγ2, anti-Vγ3, or anti-Vγ1.1 Abs. (C) Absolute numbers of fetal γδ T cell subpopulations (mean ± SEM, n = 6–12). *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. n.s., not significant.

FIGURE 6.

Rearrangements of the Jγ1 gene segment and development of γδ T cells of γ1 cluster origin are severely impaired in fetal thymus of Jγ1 promoter mutant mice. (A) Vγ-Jγ coding joints were measured by real-time PCR in Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f embryonic day 17.5 fetal thymocytes. Coding joint levels were normalized to those of genomic DNA (IL-7R), and rearrangement levels relative to that of Jγ1Pf/f mice are shown as the mean ± SEM of two to four experiments. (B) Embryonic day 17.5 thymocytes of Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f mice were stained with anti-CD3 and anti-TCRγδ, anti-Vγ2, anti-Vγ3, or anti-Vγ1.1 Abs. (C) Absolute numbers of fetal γδ T cell subpopulations (mean ± SEM, n = 6–12). *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. n.s., not significant.

Close modal

We next analyzed the development of γδ T cells in fetal thymus of Jγ1 promoter mutant mice. The number of Vγ2+ and Vγ3+ T cells was severely reduced in fetal thymus of Jγ1PΔ/Δ and Jγ1PmS/mS mice relative to controls, whereas Vγ1.1+ T cells were slightly increased (Fig. 6B, 6C). These results demonstrate that the development of γδ T cells of γ1 cluster origin is specifically impaired at the fetal stage in Jγ1 promoter mutant mice. Interestingly, defects in rearrangements and development were more severe in Jγ1PmS/mS mice than in Jγ1PΔ/Δ mice, as was the case in adult mice.

Because Vγ3+ T cells preferentially develop in fetal thymus and are distributed as DETCs in adults, we analyzed DETCs in promoter mutant and control mice using flow cytometry. The ratio of γδ T cells/Langerhans cells (I-A/I-E+) was slightly decreased in Jγ1PmS/mS mice relative to controls (Fig. 7A, 7B). Although there were slightly fewer Vγ3+ T cells in Jγ1PΔ/Δ mice, these cells were severely decreased in Jγ1PmS/mS mice. In contrast, the number of Vγ1.1+ DETCs was significantly increased in Jγ1PmS/mS mice but modestly elevated in Jγ1PΔ/Δ mice. We then characterized epidermal sheets by immunohistochemistry. Cell density and the ratio of γδ DETCs/Langerhans cells were modestly reduced in Jγ1 promoter mutant mice (Fig. 7C, Supplemental Fig. 4B). Although the number of Vγ3+ DETCs was slightly decreased in Jγ1PΔ/Δ mice, it was severely decreased in Jγ1PmS/mS mice. These results indicate that seeding of Vγ3+ T cells into the epidermis is severely impaired in Jγ1PmS/mS mice and are consistent with the observation that Vγ3-Jγ1 rearrangement is completely blocked in Jγ1PmS/mS fetal thymus.

FIGURE 7.

Vγ3+ T cells are reduced in the skin of Jγ1 promoter mutant mice. (A) Epidermal cells were isolated from Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f mice at 4–5 wk old. Cells were stained with anti-TCRγδ and anti–I-A/I-E Abs or with anti-CD3 and anti-Vγ3/Vδ1 or anti-Vγ1.1 Abs. (B) The ratio of TCRγδ+, Vγ3+, and Vγ1.1+ DETCs/Langerhans cells (I-A/I-E+) was calculated (mean ± SEM, n = 3–5). (C) Numbers of TCRγδ+ and Vγ3+ DETCs and their ratio to Langerhans cells (I-A/I-E+) were calculated from three to five epidermal sheets in Supplemental Fig. 4B (mean ± SEM, n = 2). *p < 0.05, **p < 0.01, ****p < 0.001. n.s., not significant.

FIGURE 7.

Vγ3+ T cells are reduced in the skin of Jγ1 promoter mutant mice. (A) Epidermal cells were isolated from Jγ1PΔ/Δ, Jγ1PmS/mS, and Jγ1Pf/f mice at 4–5 wk old. Cells were stained with anti-TCRγδ and anti–I-A/I-E Abs or with anti-CD3 and anti-Vγ3/Vδ1 or anti-Vγ1.1 Abs. (B) The ratio of TCRγδ+, Vγ3+, and Vγ1.1+ DETCs/Langerhans cells (I-A/I-E+) was calculated (mean ± SEM, n = 3–5). (C) Numbers of TCRγδ+ and Vγ3+ DETCs and their ratio to Langerhans cells (I-A/I-E+) were calculated from three to five epidermal sheets in Supplemental Fig. 4B (mean ± SEM, n = 2). *p < 0.05, **p < 0.01, ****p < 0.001. n.s., not significant.

Close modal

In this study, we investigated whether direct binding of STAT5 to STAT motifs in the Jγ promoters are essential for chromatin accessibility and rearrangements of Jγ gene segments. Germline transcription and active histone modifications of the Jγ1 gene segment were severely and specifically reduced in thymocytes of Jγ1PΔ/Δ and Jγ1PmS/mS promoter mutant mice. The STAT motifs also were required for a Jγ1/Eγ1 chromatin interaction. Rearrangements and DNA cleavage of the Jγ1 gene segment were dramatically diminished in mutant mice. Impaired rearrangements resulted in severely reduced numbers of γδ T cells of γ1 cluster origin at both the fetal and adult stages. These results strongly suggest that direct binding of STAT5 to the STAT motifs in a Jγ promoter is essential for triggering local chromatin accessibility and rearrangements of the Jγ gene segment in vivo.

STAT motifs found in Jγ promoters likely control local chromatin accessibility. It is reported that germline promoters regulate local chromatin accessibility of downstream gene segments in the TCRα, TCRβ, IgH, and Igκ loci (8, 9, 50). Indeed, we observed that deletion of the Jγ1 promoter or mutations in the STAT motifs found in that promoter specifically reduced accessibility and recombination of the Jγ1 gene segment. Because STAT motifs are conserved in other Jγ germline promoters, this mechanism may operate in all Jγ gene segments. In contrast, Eγ elements likely control locus-wide accessibility of the TCRγ locus. We reported that STAT5 augments the transcriptional activity of Eγ elements (20). Future studies should address whether STAT5 controls locus-wide accessibility of the TCRγ locus through binding to STAT motifs in Eγ elements.

It is reported that IL-7R signaling functions in γδ T cell development by controlling transcription of TCRγ genes rather than recombination of the TCRγ locus (51). Because the STAT motifs are deleted by Vγ-Jγ recombination, our observation that Jγ1 rearrangements are impaired in Jγ1 promoter mutant mice indicates that STAT motifs directly control Jγ1 chromatin accessibility prior to recombination. Nevertheless, our study does not exclude the possibility that STAT5 increases transcription of rearranged TCRγ genes through Eγ elements.

STAT5 is likely to recruit RAG recombinases to Jγ gene segments by several mechanisms. STAT5 triggers histone H3K4me3 modification required for RAG2 recruitment and activation (38, 52). Jγ1PΔ/Δ and Jγ1PmS/mS mice showed severe impairment in H3K4me3 modification and recombination of the Jγ1 gene segment. STAT5 also promotes an open chromatin status through germline transcription and histone modifications, an activity required for accessibility of RAG1, and perhaps RAG2, to Jγ gene segments. Germline transcription mediates chromatin accessibility by repositioning and evicting nucleosomes, and transcriptional elongation is essential for RAG recombinase accessibility at the TCRα locus (53, 54). Furthermore, the SWI/SNF complex increases RAG1/2-mediated DNA cleavage at recombination signal sequences in the absence of RNA polymerase II–mediated transcription or histone modifications (45, 55). We found that germline transcription, H3ac, H3K4 methylations, and association of the SWI/SNF complex at the Jγ1 gene segment were severely reduced in Jγ1 promoter mutant mice. Therefore, our study strongly suggests that binding of STAT5 to Jγ promoters recruits RAG1 and RAG2 recombinases to Jγ gene segments through germline transcription, active histone modifications, and recruitment of chromatin-remodeling complexes. In addition, one of the TCRγ enhancers, Eγ1, interacted with the Jγ1 promoter, and this interaction was dependent on STAT motifs in the Jγ1 promoter (Fig. 3B). However, interaction between the Jγ1 promoter and Eγ1 was not necessarily required for germline transcription of Jγ1, because Jγ1 was normally transcribed in Eγ1-deficient mice (Fig. 3C). This may be because HsA is sufficient to induce Jγ1 transcription, because a previous study showed that Eγ1 and HsA redundantly regulate Vγ-Jγ1 rearrangements (48). Alternatively, HsA, as well as other Eγ(s) (i.e., Eγ2, Eγ3, and Eγ4) or unknown cis-regulatory elements, might redundantly regulate Jγ1 transcription with Eγ1.

Recruitment of STAT5 to the Jγ promoters may be one of the earliest steps for chromatin accessibility of Jγ gene segments. H3K4me1 reportedly takes place in the priming of lineage-determining genes at the first step of cell differentiation before H3K4me2 and H3K4me3 (44, 56). Because H3K4me1 was significantly reduced at the Jγ1 gene segment in Jγ1PmS/mS mice, STAT5 binding to the Jγ1 promoter appears to be one of the earliest events in Jγ1 accessibility in early T cells. After H3K4me1, the H3K4me2 mark is induced prior to histone acetylation by histone acetyltransferases (21), followed by chromatin remodeling by the SWI/SNF complex and transcription by RNA polymerase II (57). H3K4me3 is tightly linked with RNA polymerase II activity and transcription (44). Histone-acetylated nucleosomes stabilize binding of the SWI/SNF complex and enhance its remodeling activity (58, 59). In contrast, the SWI/SNF complex also increases histone acetylation (55). Taken together, these histone modifications and associations of histone acetyltransferases, the SWI/SNF complex, and RNA polymerase II cooperatively may induce fully accessible recombination signal sequences after STAT5 recruitment in the TCRγ locus.

Jγ1PmS/mS mice showed more severe impairment in chromatin accessibility and rearrangements of the Jγ1 gene segment than did Jγ1PΔ/Δ mice. Specifically, germline transcription was reduced by >2700-fold in adult Jγ1PmS/mS mice compared with >600-fold in Jγ1PΔ/Δ mice. Vγ5-Jγ1 coding joints were decreased 61- and 33-fold and Vγ2-Jγ1 coding joints were reduced 215- and 84-fold in adult Jγ1PmS/mS and Jγ1PΔ/Δ immature thymocytes, respectively. Similar results were obtained with coding joints in fetal thymocytes of Jγ1PmS/mS and Jγ1PΔ/Δ mice. In addition, Vγ3+ T cells in fetal thymus and adult epidermis were reduced more severely in Jγ1PmS/mS mice compared with Jγ1PΔ/Δ mice. It is not clear what accounts for these differences in phenotypic severity. Possibly, a cryptic upstream promoter activates weak transcriptional activity in Jγ1PΔ/Δ mice in the absence of the Jγ1 promoter. Alternatively, some repressive sequences within the Jγ1 promoter of Jγ1PmS/mS mice may recruit negative regulators that could be displaced by STAT5 binding in Jγ1Pf/f mice.

Jγ1 promoter mutant lines manifested contrasting phenotypes relevant to Vγ3+ DETCs. Although both Jγ1PΔ/Δ and Jγ1PmS/mS mice showed severely impaired rearrangements and development of Vγ3+ T cells in fetal thymus, only Jγ1PmS/mS mice showed significantly reduced numbers of Vγ3+ DETCs. This difference might be explained if a few Vγ3+ γδ T cells developed in fetal thymus of Jγ1PΔ/Δ mice and then expanded extensively in the epidermis. In contrast, Vγ3-Jγ1 recombination was completely blocked in Jγ1PmS/mS mice, resulting in severely reduced numbers of Vγ3+ DETCs. These results are consistent with a previous report that Vγ3+ T cells can expand extensively in the epidermis (60). In addition, the increase in the number of Vγ1.1+ DETCs in Jγ1 promoter mutant mice is reminiscent of similar results reported in Vγ3-disrupted mice (61).

V(D)J recombination is controlled by local chromatin accessibility, as well as by higher-order chromatin configuration, such as chromatin loop, locus compaction, and subnuclear localization of AgR loci (62). However, molecular mechanisms controlling higher-order chromatin configuration remain largely unexplored. STAT5 activated by IL-7R reportedly regulates distal VH gene rearrangements but is dispensable for nuclear repositioning or compaction (11). The present study clearly showed that Jγ1/Eγ1 interaction within the γ1 cluster was controlled by STAT motifs in the Jγ1 promoter. It will be of interest to use Jγ1 promoter mutant mice to determine whether STAT5 triggers chromatin loop formation between Jγ promoters and other Eγ elements and whether compaction or subnuclear localization of the TCRγ locus is induced by STAT5.

In summary, this study strongly suggests that direct binding of STAT5 to STAT motifs in the Jγ1 promoter is essential for triggering local chromatin accessibility, Jγ1/Eγ1 interaction, and rearrangements of the Jγ1 gene segment. Considering conservation of STAT motifs in Jγ germline promoters and Eγ enhancers, STAT5 likely regulates the entire TCRγ locus through these motifs. Our Jγ1 promoter mutant mice should accelerate efforts to dissect molecular mechanisms underlying local and locus-wide control of the TCRγ locus by STAT5.

We thank Drs. J. Takeda, G. Kondoh, and K. Yusa for the KY1.1 ES line and targeting system; Dr. N.G. Copeland for the Red recombination system; Drs. I. Saito, Y. Shinkai, M. Tachibana, and Y. Matsumura for AxCAFLP and AdV-Cre adenoviruses; Dr. H. Karasuyama for J558–IL-7 cell line; M. Kishida, Dr. K. Imai, H. Hayashi, and S. Kamioka for excellent technical assistance; Dr. Y. Agata for encouragement; and members of the Ikuta Laboratory for discussion.

This work was supported by Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) Grants-in-Aid for Scientific Research (C) 25460589 and Scientific Research on Innovative Areas 25111504 (to K. Ikuta), Young Scientists (B) 24790469 and Scientific Research (C) 26460572 (to S.T.-i.), and Young Scientists (B) 24790468 (to T.H.), as well as by a MEXT funded Global Centers of Excellence Program “Center for Frontier Medicine.” This work was also supported by grants from the Fujiwara Memorial Foundation and the Shimizu Foundation for Immunology and Neuroscience, as well as a BioLegend/Tomy Digital Biology Young Scientist Research Award for 2011.

The online version of this article contains supplemental material.

Abbreviations used in this article:

BAC

bacterial artificial chromosome

3C

chromosome conformation capture

ChIP

chromatin immunoprecipitation

DETC

dendritic epidermal T cell

TCRγ 3′ enhancer

ES

embryonic stem

H3ac

histone H3 acetylation

H3K4

histone H3 lysine 4

H3K4me1

H3K4 monomethylation

H3K4me2

H3K4 dimethylation

H3K4me3

H3K4 trimethylation

HsA

DNase I hypersensitivity site

IEL

intraepithelial lymphocyte

LM

ligation mediated

TN

CD3CD4CD8 triple negative.

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The authors have no financial conflicts of interest.

Supplementary data