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Cutting Edge: HDAC3 Protects Double-Positive Thymocytes from P2X7 Receptor–Induced Cell Death

Rachael L. Philips, Shaylene A. McCue, Matthew J. Rajcula and Virginia S. Shapiro
J Immunol February 15, 2019, 202 (4) 1033-1038; DOI: https://doi.org/10.4049/jimmunol.1801438
Rachael L. Philips
Department of Immunology, Mayo Clinic, Rochester, MN 59905
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Shaylene A. McCue
Department of Immunology, Mayo Clinic, Rochester, MN 59905
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Matthew J. Rajcula
Department of Immunology, Mayo Clinic, Rochester, MN 59905
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Virginia S. Shapiro
Department of Immunology, Mayo Clinic, Rochester, MN 59905
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Abstract

Intricate life-versus-death decisions are programmed during T cell development, and the regulatory mechanisms that coordinate their activation and repression are still under investigation. In this study, HDAC3-deficient double-positive (DP) thymocytes exhibit a severe decrease in numbers. The thymic cortex is rich in ATP, which is released by macrophages that clear apoptotic DP thymocytes that fail to undergo positive selection. We demonstrate that HDAC3 is required to repress expression of the purinergic receptor P2X7 to prevent DP cell death. HDAC3-deficient DP thymocytes upregulate the P2X7 receptor, increasing sensitivity to ATP-induced cell death. P2rx7/HDAC3-double knockout mice show a partial rescue in DP cell number. HDAC3 directly binds to the P2rx7 enhancer, which is hyperacetylated in the absence of HDAC3. In addition, RORγt binds to the P2rx7 enhancer and promotes P2X7 receptor expression in the absence of HDAC3. Therefore, HDAC3 is a critical regulator of DP thymocyte survival and is required to suppress P2X7 receptor expression.

Introduction

The accurate coordination of transcriptional regulators, chromatin modifiers, and nucleosome remodelers is critical for proper gene expression during T cell development (1). Changes in gene expression occur as thymocytes transition through multiple checkpoints, resulting in different cellular fates. Cell death is a common fate decision, as the majority of thymocytes [especially double-positive (DP) thymocytes (2)] fail to complete T cell development. Specifically, TCR signaling controls the fate of DP thymocytes, as an absence of TCR signaling leads to death by neglect, a strong TCR signal leads to death by negative selection, or a weak TCR signal leads to thymocyte survival via positive selection (reviewed in Ref. 3). Each of these cellular fates is coordinated by the activation or repression of different transcriptional programs.

Gene repression is as important as gene activation at each stage of thymocyte development. At the chromatin level, gene repression works through the recruitment of corepressor complexes. Histone deacetylase (HDAC) 3 is a corepressor important in immune cell development (reviewed in Ref. 4). HDAC3 belongs to the class I family of HDACs and functions to remove acetyl groups from both histone tails and nonhistone proteins (5). HDAC3 acts as the catalytic component of the N-CoR corepressor complex and facilitates gene repression through its recruitment to promoters or enhancers (6). The HDAC3/N-CoR complex does not have a DNA-binding domain, therefore it must be recruited to specific sites in the genome via its interaction with different transcription factors.

Previously, HDAC3 was shown to be critical for thymocyte positive selection (7, 8). When HDAC3 is conditionally deleted in thymocytes using CD2-icre (named HDAC3–conditional knockout [cKO] mice hereafter), there is a marked reduction in the number of DP and single-positive (SP) thymocytes (7). The reduction in SP thymocytes is due to a block in positive selection and could not be rescued by an OT-II TCR transgene. OT-II HDAC3-cKO mice also exhibit a positive selection block, comparable to HDAC3-cKO mice. Mechanistically, HDAC3 is required to repress retinoic acid–related orphan receptor (ROR)γt during positive selection, as RORγt is normally downregulated at this stage, and constitutive expression of RORγt leads to a similar block in positive selection (9). Deletion of RORγt, in conjunction with a Bcl-xl transgene [necessitated by the dependence of Bcl-xl expression on RORγt (10)], alleviated the block in positive selection resulting from HDAC3 deficiency (RORγt-KO Bcl-xl transgenic [Tg] HDAC3-cKO, hereafter called “RB3”). In addition, DP cellularity was restored in RB3 mice, although the mechanism was not known. The focus of this paper is the mechanism by which HDAC3 regulates DP thymocyte survival.

In this study, we find one cause for the survival defect in DP thymocytes from HDAC3-cKO mice. HDAC3-deficient DP thymocytes exhibit increased expression of the purinergic receptor P2X7 (encoded by the P2rx7 gene). Cells that express P2X7 receptor are more sensitive to high concentrations of extracellular ATP, which results in large pore formation and loss of membrane integrity (reviewed in Ref. 11). The regulation of P2rx7 expression is coordinated by HDAC3 and RORγt at the P2rx7 enhancer. HDAC3 deletion leads to an increase in histone acetylation at the P2rx7 gene locus and deletion of RORγt normalizes P2X7 receptor expression in HDAC3-deficient DP thymocytes. Therefore, HDAC3 is required to suppress P2X7 receptor expression in DP thymocytes and promote DP survival.

Materials and Methods

Mice

HDAC3 fl/fl mice were provided by S. Hiebert [Vanderbilt (12)]. Human Bcl-2 Tg mice were generated by S. Korsmeyer (13) and provided by A. Singer (National Institutes of Health). Bcl-xL Tg mice (14), RORγt -KO mice (15), CD2-icre mice (16), and P2rx7-KO (17, 18) mice were purchased from The Jackson Laboratory. OT-II (19) mice were purchased from Taconic Biosciences. Mice were housed in barrier facilities, and experiments were performed at the Mayo Clinic with Institutional Animal Care and Use Committee approval. Mice were analyzed between the ages of 4 and 8 wk with either littermates or age-matched controls (termed wild-type [WT]), which may include floxed-only mice (no Cre), CD2-icre, or WT mice, as no differences were observed between these mice.

Flow cytometry

FACS analysis was performed on an Attune NxT Flow Cytometer (Thermo Fisher Scientific) and analyzed with FlowJo (Tree Star). Experiments were acquired live or fixed (BD Cytofix/Cytoperm Fixation and Permeabilization Kit; BD Biosciences). Bcl-xl staining used the Foxp3/Transcription Factor Staining Buffer Kit (Tonbo Biosciences). All analyses included size exclusion (forward scatter [FSC] area/side scatter area), doublet exclusion (FSC height/FSC area), and dead cell exclusion (Ghost Dye Red 780; Tonbo Biosciences). Abs used were: CD4 (GK1.5 or RM4-5), CD8α (53-6.7 or 2.43), CD11b (M1/70), CD45.2 (104), CD45.1 (A20), Bcl-xl (7B2.5), P2X7 receptor (polyclonal; Enzo Life Sciences), RORγt (AFKJS-9), B220 (RA3-62B), CD19 (6D5), CD11c (N418), NK1.1 (PK136), Gr-1 (RB6-8C5), Ter119 (TER-119), and TCRβ (Η57−597).

Bone marrow mixed chimeras

Mixed bone marrow chimeras were generated by i.v. injection of 4 × 106 cells from 50:50 mixes of either WT (CD45.2+)/B6.SJL (CD45.1+) or CD2-icre HDAC3-cKO (CD45.2+)/B6.SJL (CD45.1+) mice into lethally irradiated congenic B6.SJL (CD45.1+) recipients. Mice received enrofloxacin in their drinking water for 3 wk and were analyzed after 8 wk.

Ex vivo stimulation

Thymocytes were cultured at 4 × 106 cells/ml with/without 1 mM ATP (Sigma) or 100 μM 2′(3′)-O-(4-benzoylbenzoyl) ATP (BzATP; Tocris Bioscience) in culture medium (RPMI 1640, 10% FCS, penicillin/streptomycin/glutamine). For experiments using A438079 (Abcam), cells were pretreated with 10 or 100 μM A438079 for 1 h before addition of ATP or BzATP. After 15 min of stimulation, cells were harvested, washed, stained for Annexin V binding (Apoptosis Detection kit; BD Biosciences). To measure pore formation, 2 μM 4-[(3-methyl-1,3-benzoxazol-2(3H)-ylidene)methyl)]-1-[3-(trimethylammonio)propyl] quinolinium diiodide (YO-PRO-1) was added to the culture for the last 5 min before harvesting, washing, and staining.

Downloaded datasets

The following datasets were retrieved from Gene Expression Omnibus series: GSM726991 (RNA polymerase II), GSM1556287 (acetylated histone H3 lysine-27 [H3K27ac]), GSM726994 (trimethylated histone H3 lysine-4), GSM945565 (trimethylated histone H3 lysine-27), GSM726993 (monomethylated histone H3 lysine-4 [H3K4me1]), GSE63731 (capturing self-transcribing active regulatory region sequencing [CapSTARR-seq]), and GSM2354271 (RORγt). Sequencing data were imaged using the Integrative Genomics Viewer software (Broad Institute).

Quantitative chromatin immunoprecipitation

For quantitative chromatin immunoprecipitation (qChIP), DP thymocytes were enriched using the EasySep Mouse Streptavidin RapidSpheres Isolation Kit (#19860; STEMCELL Technologies) to remove SP, double-negative (DN), and γδ thymocytes with biotin-conjugated anti-CCR7 (4B12), anti-IL-7Rα (A7R34), anti-H2K (AF6-88.5), anti-CD44 (IM7), anti-CD25 (PC61.5), and anti-TCRγδ (UC7-13D5). DP thymocyte purity was at least 95%. Cells were fixed with 1% formaldehyde for 10 min (H3K27ac qChIP) or 15 min (HDAC3 qChIP) and quenched with 125 mM glycine for 10 min. H3K27ac ChIP was performed according to (20) using anti-H3K27ac (#ab4729; Abcam). HDAC3 ChIP was performed according to (21), using anti-HDAC3 (#85057; Cell Signaling Technology), with the following adjustments: after cell lysis and brief sonication (4 min, 30 s on/30 s off; Bioruptor Pico; Diagenode), an equal volume of 2× MNase buffer (35 mM Tris-HCl [pH 7.5], 25 mM NaCl, 120 mM KCl, 2 mM CaCl2) was added to each sonication sample and digested with MNase (#10011S; Cell Signaling Technology) at 37°C for 15 min. Isolated DNA was used to perform real-time PCR. Graphs depict fold enrichment to regions without H3K27ac (Intergenic primers) or HDAC3 binding (Rpl30 primers). Primers used for H3K27ac qChIP are as follows: P2rx7 enhancer forward, 5′-GGTGGGGTGACGAAGTTAGG-3′; P2rx7 enhancer reverse, 5′-GAATTCCACGGCACTCACCT-3′; Intergenic forward, 5′-CCTGCTGCCTTGTCTCTCTC-3′; and Intergenic reverse, 5′-ATGGCCTAGGGATTCCAGCA-3′. Primers used for HDAC3 qChIP are: P2rx7 promoter forward, 5′-AGACTGTGTGCCTCCCTTTG-3′; P2rx7 promoter reverse, 5′-CCCTTATCTCTGTGGGAGCC-3′; P2rx7 enhancer forward, 5′-GAACAGTTCCTGCGGCTTTG-3′; P2rx7 enhancer reverse, 5′-CTTTTGAAACCAGCCGTGGG-3′; and Rpl30 purchased from Cell Signaling Technology (#7015).

Statistical analysis

Two-tailed unpaired Student t test (GraphPad Prism) was used to compare groups. Boxes on boxplots encompass the 25th to 75th percentile, and whiskers extend to the minimum and maximum values.

Results and Discussion

Decreased viability of HDAC3-deficient DP thymocytes is cell intrinsic and not rescued by Bcl-xl or Bcl-2 transgenes

HDAC3-cKO mice had ∼80% fewer DP thymocytes as compared with WT mice [(7), Fig. 1A]. The reduction was unlikely to be due to the positive selection block in HDAC3-cKO mice because mice that lack TCR-α, MHC, or key TCR signaling molecules (e.g., Zap70) do not have compromised DP thymocyte numbers (22–24). To determine whether the DP reduction was cell intrinsic, 50:50 mixed bone marrow chimeric mice were generated. Chimerism frequency was measured at the DN-to-DP stages, with splenic CD11b+ cells used as a control. Chimerism frequency was slightly reduced in HDAC3-deficient DN4 and immature CD8 single-positive thymocytes, whereas HDAC3-deficient DP thymocytes exhibited a large reduction in chimerism (Fig. 1B), demonstrating that the effect is cell intrinsic. Although HDAC3 protein deletion starts at DN3 in HDAC3-cKO mice, there was not a deficiency in DN cellularity, β selection, and proliferation in DN3 and DN4 thymocytes (7). Therefore, it is unlikely that the deficit in DP cell number is due to a defect prior to the DP stage.

FIGURE 1.
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FIGURE 1.

HDAC3-deficient DP thymocytes exhibit a Bcl-xl/Bcl-2–independent survival defect. (A) Representative FACS plots of DP thymocytes from WT and HDAC3-cKO mice. Boxplots show DP cellularity with four mice per group. (B) 50:50 mixed bone marrow chimeras of B6.SJL (CD45.1+) bone marrow mixed with WT (CD45.2+) or HDAC3-cKO (CD45.2+). Plots depict mean ± SEM percent chimerism (n = 5 per group) of splenic CD11b+ cells as well as DN and DP thymocytes between CD45.1+ and CD45.2+ cells. DN2-DN4 thymocytes were identified as kit+CD25+ (DN2), c-kit−CD25+ (DN3), and c-kit−CD25− (DN4) after gating from CD3− lineage− (B220/CD19, CD11b, CD11c, NK1.1, Gr-1, Ter119, CD4, CD8, TCRβ). (C) Representative FACS plot depicting Bcl-xl expression in DP thymocytes from three WT and three HDAC3-cKO mice. (D) Number of DP thymocytes from WT, HDAC3-cKO, Bcl-xl tg, Bcl-xl tg/HDAC3-cKO, Bcl-2 tg, and Bcl-2 tg/HDAC3-cKO mice. Expression of both Bcl-xl and Bcl-2 transgenes are driven by the proximal Lck promoter. Plots show four to eight mice per group from four independent experiments.

Bcl-xl is required for DP cell survival (25); however, Bcl-xl protein expression in DP thymocytes was similar between WT and HDAC3-cKO mice (Fig. 1C). To determine whether overexpression of the Bcl-2 family anti-apoptotic protein Bcl-xl or Bcl-2 could rescue DP cell number from HDAC3-cKO mice, Bcl-xl and Bcl-2 transgenes were introduced. However, no increase in DP cell number from Bcl-xl Tg/HDAC3-cKO mice or Bcl-2 Tg/HDAC3-cKO mice was observed as compared with HDAC3-cKO mice (Fig. 1D). Thus, the DP survival defect in HDAC3-cKO mice cannot be compensated by overexpression of Bcl-xl or Bcl-2 (7).

HDAC3-deficient DP thymocytes are susceptible to P2X7 receptor–induced cell death

The purinergic receptor P2X7 induces thymocyte cell death upon stimulation with high doses of ATP (26). The thymic cortex is believed to be an ATP-rich environment, as resident macrophages release ATP as a result of phagocytosing DP thymocytes undergoing cell death (27). WT DP thymocytes express low levels of P2X7 receptor compared with DN and SP thymocytes and are thus relatively insensitive to extracellular ATP-induced cell death (Fig. 2A, Supplemental Fig. 1). However, HDAC3-deficient DP thymocytes significantly upregulated P2X7 receptor as compared with WT DP thymocytes (Fig. 2A) and were more sensitive to P2X7 receptor–induced cell death from an ex vivo culture with the P2X7 receptor ligand ATP or the P2X7 receptor agonist BzATP (Fig. 2B). Preincubation of HDAC3-deficient thymocytes with the P2X7 receptor–specific antagonist A438079 abrogated the increase in Annexin V binding caused by ATP treatment (Fig. 2C), demonstrating that the ATP-induced cell death was not due to stimulation of other purinergic receptors coexpressed by HDAC3-deficient DP thymocytes. HDAC3-deficient DP thymocytes required a higher dose of A438079 to abrogate BzATP-induced Annexin V staining (Fig. 2C), again suggesting that HDAC3-deficient DP thymocytes are more sensitive to P2X7 receptor ligands. Strong stimulation of P2X7 receptor can also induce cell membrane pore-mediated cell death (28). YO-PRO-1 is a large (∼600 Da) nucleic acid stain that labels cells with compromised plasma membranes and is therefore a surrogate marker for pore formation (29). After 1 h stimulation of thymocytes with ATP or BzATP, HDAC3-deficient DP thymocytes exhibited an increase in YO-PRO-1 staining compared with unstimulated, etoposide stimulation, or WT controls (Fig. 2D, Supplemental Fig. 2), demonstrating that P2X7 receptor–induced cell death in HDAC3-cKO mice occurs via pore formation.

FIGURE 2.
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FIGURE 2.

HDAC3-deficient DP thymocytes are susceptible to cell death mediated by the P2X7 receptor. (A) P2X7 receptor expression on DP thymocytes from five WT and seven HDAC3-cKO mice from three independent experiments. (B and C) Frequency of Annexin V+ DP thymocytes stimulated for 15 min ex vivo with 1 mM of ATP or 100 μM of BzATP from WT and HDAC3-cKO mice, with or without a 1-h pretreatment with the P2X7 receptor antagonist A438079. Plots show mean ± SEM of three to four mice per group from three independent experiments. (D) Frequency of YO-PRO-1+ DP thymocytes, from WT and HDAC3-cKO mice, stimulated for 1 h ex vivo with 1 mM ATP or 100 μM BzATP. Data are representative of three to four mice from three independent experiments. (E and F) Frequency of DP thymocytes that are Annexin V+ (E) or YO-PRO-1+ (F) after ex vivo stimulation with 1 mM of ATP or 100 μM of BzATP for 15 min or 1 h, respectively. DP thymocytes are from WT, HDAC3-cKO, P2rx7-KO, or P2rx7/HDAC3-DKO mice. Plot shows mean ± SEM of three to four mice from three independent experiments. (G) Number of DP thymocytes from WT, HDAC3-cKO, P2rx7-KO, or P2rx7/HDAC3-DKO mice. Boxplots depict four to five mice from four independent experiments.

To understand the contribution of the P2X7 receptor to reduced DP cell survival in HDAC3-cKO mice, P2rx7/HDAC3–double knockout (DKO) mice were generated. Loss of the P2X7 receptor protected HDAC3-deficient DP thymocytes from ATP- and BzATP-induced cell death (Fig. 2E). Similarly, knocking out P2rx7 abrogated the increase in YO-PRO-1 staining in response to either ATP or BzATP in P2rx7/HDAC3-DKO mice compared with HDAC3-cKO mice (Fig. 2F), demonstrating that pore formation is specifically induced by the P2X7 receptor. Examination of DP cell number from P2rx7/HDAC3-DKO mice revealed a 2-fold increase in cell number compared with HDAC3-cKO mice (Fig. 2G); however, the number of DP thymocytes from P2rx7/HDAC3-DKO mice was still below WT mice (Fig. 2G). This indicates that there must be other causes of DP cell death in addition to increased expression of P2X7.

The P2rx7 gene locus is suppressed by HDAC3 in DP thymocytes

Examination of P2X7 receptor expression during T cell develop revealed that P2rx7 is specifically downregulated at DP stage compared with DN and CD4SP thymocytes (Supplemental Fig. 1). Because deletion of HDAC3 in DP thymocytes leads to P2X7 receptor upregulation (Fig. 2A), publicly available genome sequencing datasets were used to examine the chromatin state of the P2rx7 gene locus in WT thymocytes. Cd8a and Hoxc4 gene loci were used as controls for highly expressed genes and repressed genes in thymocytes, respectively. Compared with Cd8a, the P2rx7 locus showed a low signal for RNA polymerase II, H3K27ac, and trimethylated histone H3 lysine-4 (Fig. 3A), indicating that the P2rx7 locus does not show chromatin marks of active gene expression in WT thymocytes. This is consistent with low P2X7 receptor expression in WT thymocytes (Fig. 2A). Interestingly, the repressive mark trimethylated histone H3 lysine-27 was not enriched at the P2rx7 locus (Fig. 3A), suggesting that P2rx7 is not actively repressed by a polycomb repressive complex–regulated mechanism. In addition, H3K4me1 ChIP-seq and CapSTARR-seq were used to identify enhancers in WT thymocytes and examine their activity (30, 31), respectively. Within intron 2 of P2rx7, an enhancer was revealed by enrichment of H3K4me1 (Fig. 3A), which is consistent with previous reports (32). The combination of H3K27ac and H3K4me1 marks identifies active enhancers (30); however, the P2rx7 enhancer lacked H3K27ac (Fig. 3A), indicating that the P2rx7 enhancer is not active in WT thymocytes. To validate P2rx7 enhancer activity in thymocytes, we used publicly available CapSTARR-seq (31). Consistent with the absence of H3K27ac at this enhancer, the CapSTARR-seq signal was also absent at the P2rx7 enhancer (Fig. 3A), confirming that the enhancer is inactive in WT DP thymocytes. Therefore, the P2rx7 gene locus is suppressed in WT thymocytes.

FIGURE 3.
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FIGURE 3.

The P2rx7 gene locus is repressed by HDAC3 in DP thymocytes. (A) ChIP-seq and CapSTARR-seq snapshots at P2rx7, Cd8a, and Hoxc4. Yellow boxes identify previously characterized promoter (P) and enhancer (E) regions (32). (B) HDAC3 qChIP at the P2rx7 promoter and enhancer in DP thymocytes from WT and HDAC3-cKO mice. Plots show mean ± SEM of three to four mice per group from three independent experiments. (C) H3K27ac qChIP at the P2rx7 enhancer in DP thymocytes from WT and HDAC3-cKO mice. Plots show mean ± SEM of three mice per group from three independent experiments.

HDAC3 regulates gene expression upon recruitment to gene promoters or enhancers. To determine whether HDAC3 binds to either of these regions of the P2rx7 gene, HDAC3 qChIP was performed on DP thymocytes from WT mice, with HDAC3-cKO mice used as a negative control for HDAC3 binding. HDAC3 qChIP revealed that HDAC3 bound to the P2rx7 enhancer but not the P2rx7 promoter (Fig. 3B), indicating that HDAC3 directly regulates P2rx7 expression. Because HDAC3 deacetylates histones, H3K27Ac was analyzed at the P2rx7 enhancer by qChIP in WT and HDAC3-deficient DP thymocytes. Whereas DP thymocytes from WT mice exhibited low levels of acetylation at the P2rx7 enhancer (similar to Fig. 3A), deletion of HDAC3 increased acetylation at the P2rx7 enhancer in DP thymocytes from HDAC3-cKO mice (Fig. 3C), demonstrating HDAC3 directly regulates histone acetylation at the P2rx7 gene locus.

RORγt promotes P2X7 receptor expression in HDAC3-deficient DP thymocytes

HDAC3 is required to repress RORγt during positive selection (7), as RORγt is normally downregulated at this stage, and constitutive expression of RORγt leads to a similar block in positive selection, as observed in HDAC3-cKO mice (9). Deletion of RORγt rescues the block in positive selection in RB3 mice as well as DP cellularity (7), suggesting that RORγt may regulate P2X7 receptor expression. A previous study identified retinoic acid response elements in the P2rx7 intronic enhancer and RARα binding to this enhancer in CD4+ T cells (32). RORγt belongs to the ROR family of transcription factors that show sequence homology to retinoic-acid receptor family of proteins (33). Therefore, RORγt may bind to the P2rx7 enhancer in WT thymocytes. Publicly available RORγt ChIP-seq dataset of WT thymocytes demonstrates that RORγt associates with the P2rx7 enhancer in WT thymocytes (Fig. 4A). HDAC3 does not have a DNA binding domain, therefore HDAC3 must be recruited by transcription factors to perform its repressive function. RORγt may function to recruit HDAC3 to the P2rx7 enhancer. To test this, HDAC3 qChIP was performed in RORγt-KO Bcl-xl tg DP thymocytes, with the Bcl-xl transgene used to compensate for reduction in the number of DP thymocytes produced by RORγt deficiency (10). Interestingly, HDAC3 binding still occurred at the P2rx7 enhancer in RORγt-deficient DP thymocytes (Fig. 4B), demonstrating that HDAC3 is not recruited to the P2rx7 enhancer via RORγt.

FIGURE 4.
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FIGURE 4.

RORγt promotes P2X7 receptor expression in HDAC3-deficient DP thymocytes. (A) RORγt ChIP-seq snapshot at P2rx7 in WT thymocytes. (B) HDAC3 qChIP at the P2rx7 enhancer in DP thymocytes from WT, HDAC3-cKO, and RORγt -KO Bcl-xl tg mice. Plot shows mean ± SEM of two to three mice per group. (C) Frequency of P2X7 receptor+ DP thymocytes from OT-II, OT-II HDAC3-cKO, OT-II HDAC3-cKO RORγt-het, and OT-II RB3 mice. Boxplot depicts four to five mice from four independent experiments.

To determine whether RORγt regulates P2rx7 expression, P2X7 receptor expression in RB3 and HDAC3-deficient DP thymocytes was examined. In these experiments, RB3 and HDAC3-cKO mice contained the OT-II transgene. OT-II HDAC3-cKO DP thymocytes exhibited an increased in the frequency of P2X7 receptor positive cells compared with OT-II thymocytes (Fig. 4C), consistent with results in HDAC3-cKO and WT DP thymocytes (Fig. 2A). Loss of RORγt expression in OT-II RB3 mice restored P2X7 receptor expression to levels comparable to WT mice (Fig. 4C). Interestingly, mice with heterozygous RORγt deficiency (OT-II RORγt-het HDAC3-cKO mice) showed an intermediate frequency of P2X7-positive cells, demonstrating that the frequency of P2X7-positive cells is exquisitely sensitive to RORγt expression (Fig. 4C). Thus, RORγt promotes P2rx7 expression in HDAC3-deficient DP thymocytes.

In summary, we have identified a novel role for HDAC3 in DP thymocytes. We demonstrate that HDAC3 is required to repress expression of the purinergic receptor P2X7 to prevent DP cell death. HDAC3-deficient DP thymocytes upregulate the P2X7 receptor, increasing sensitivity to ATP-induced cell death. P2rx7/HDAC3-DKO mice show a partial restoration in DP cell number, with twice as many DP thymocytes as HDAC3-cKO mice. Mechanistically, HDAC3 directly binds to the P2rx7 enhancer, which is hyperacetylated in the absence of HDAC3. In addition, RORγt binds to the P2rx7 enhancer and promotes P2X7 receptor expression in HDAC3-deficient DP thymocytes (model in Supplemental Fig. 3). Therefore, HDAC3 is a critical regulator of DP thymocyte survival and is required to suppress P2rx7 expression.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Michael Shapiro for thoughtful discussions and critical reading of the manuscript. We also thank the Epigenomics Development Lab (Mayo Clinic) for allowing us to use their Diagenode Sonicator.

Footnotes

  • This work was supported by National Institutes of Health Grants R56 AI122746 and T32 AI007425, the Center for Biomedical Discovery at Mayo Clinic, Mayo Graduate School funds (to R.L.P.), and Mayo Foundation funds (to V.S.S.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    BzATP
    2′(3′)-O-(4-benzoylbenzoyl) ATP
    CapSTARR-seq
    capturing self-transcribing active regulatory region sequencing
    cKO
    conditional knockout
    DKO
    double knockout
    DN
    double-negative
    DP
    double-positive
    FSC
    forward scatter
    HDAC
    histone deacetylase
    H3K27ac
    acetylated histone H3 lysine-27
    H3K4me1
    monomethylated histone H3 lysine-4
    qChIP
    quantitative chromatin immunoprecipitation
    RB3
    RORγt-KO Bcl-xL Tg HDAC3-cKO
    ROR
    retinoic acid–related orphan receptor
    SP
    single-positive
    Tg
    transgenic
    WT
    wild-type
    YO-PRO-1
    4-[(3-methyl-1,3-benzoxazol-2(3H)-ylidene)methyl]-1-[3-(trimethylammonio)propyl] quinolinium diiodide.

  • Received October 30, 2018.
  • Accepted December 10, 2018.
  • Copyright © 2019 by The American Association of Immunologists, Inc.

References

  1. ↵
    1. Shapiro, M. J.,
    2. V. S. Shapiro
    . 2011. Transcriptional repressors, corepressors and chromatin modifying enzymes in T cell development. Cytokine 53: 271–281.
    OpenUrlPubMed
  2. ↵
    1. Scollay, R. G.,
    2. E. C. Butcher,
    3. I. L. Weissman
    . 1980. Thymus cell migration. Quantitative aspects of cellular traffic from the thymus to the periphery in mice. Eur. J. Immunol. 10: 210–218.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Hogquist, K. A.,
    2. T. A. Baldwin,
    3. S. C. Jameson
    . 2005. Central tolerance: learning self-control in the thymus. Nat. Rev. Immunol. 5: 772–782.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Ellmeier, W.,
    2. C. Seiser
    . 2018. Histone deacetylase function in CD4+ T cells. Nat. Rev. Immunol. 18: 617–634.
    OpenUrl
  5. ↵
    1. Emiliani, S.,
    2. W. Fischle,
    3. C. Van Lint,
    4. Y. Al-Abed,
    5. E. Verdin
    . 1998. Characterization of a human RPD3 ortholog, HDAC3. Proc. Natl. Acad. Sci. USA 95: 2795–2800.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Guenther, M. G.,
    2. O. Barak,
    3. M. A. Lazar
    . 2001. The SMRT and N-CoR corepressors are activating cofactors for histone deacetylase 3. Mol. Cell. Biol. 21: 6091–6101.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Philips, R. L.,
    2. M. W. Chen,
    3. D. C. McWilliams,
    4. P. J. Belmonte,
    5. M. M. Constans,
    6. V. S. Shapiro
    . 2016. HDAC3 is required for the downregulation of RORγt during thymocyte positive selection. J. Immunol. 197: 541–554.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Stengel, K. R.,
    2. Y. Zhao,
    3. N. J. Klus,
    4. J. F. Kaiser,
    5. L. E. Gordy,
    6. S. Joyce,
    7. S. W. Hiebert,
    8. A. R. Summers
    . 2015. Histone deacetylase 3 is required for efficient T cell development. Mol. Cell. Biol. 35: 3854–3865.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. He, Y. W.,
    2. C. Beers,
    3. M. L. Deftos,
    4. E. W. Ojala,
    5. K. A. Forbush,
    6. M. J. Bevan
    . 2000. Down-regulation of the orphan nuclear receptor ROR gamma t is essential for T lymphocyte maturation. J. Immunol. 164: 5668–5674.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Sun, Z.,
    2. D. Unutmaz,
    3. Y. R. Zou,
    4. M. J. Sunshine,
    5. A. Pierani,
    6. S. Brenner-Morton,
    7. R. E. Mebius,
    8. D. R. Littman
    . 2000. Requirement for RORgamma in thymocyte survival and lymphoid organ development. Science 288: 2369–2373.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Di Virgilio, F.,
    2. A. L. Giuliani,
    3. V. Vultaggio-Poma,
    4. S. Falzoni,
    5. A. C. Sarti
    . 2018. Non-nucleotide agonists triggering P2X7 receptor activation and pore formation. Front. Pharmacol. 9: 39.
    OpenUrl
  12. ↵
    1. Knutson, S. K.,
    2. B. J. Chyla,
    3. J. M. Amann,
    4. S. Bhaskara,
    5. S. S. Huppert,
    6. S. W. Hiebert
    . 2008. Liver-specific deletion of histone deacetylase 3 disrupts metabolic transcriptional networks. EMBO J. 27: 1017–1028.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Sentman, C. L.,
    2. J. R. Shutter,
    3. D. Hockenbery,
    4. O. Kanagawa,
    5. S. J. Korsmeyer
    . 1991. bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell 67: 879–888.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Chao, D. T.,
    2. G. P. Linette,
    3. L. H. Boise,
    4. L. S. White,
    5. C. B. Thompson,
    6. S. J. Korsmeyer
    . 1995. Bcl-XL and Bcl-2 repress a common pathway of cell death. J. Exp. Med. 182: 821–828.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Ivanov, I. I.,
    2. B. S. McKenzie,
    3. L. Zhou,
    4. C. E. Tadokoro,
    5. A. Lepelley,
    6. J. J. Lafaille,
    7. D. J. Cua,
    8. D. R. Littman
    . 2006. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126: 1121–1133.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Zhumabekov, T.,
    2. P. Corbella,
    3. M. Tolaini,
    4. D. Kioussis
    . 1995. Improved version of a human CD2 minigene based vector for T cell-specific expression in transgenic mice. J. Immunol. Methods 185: 133–140.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Borges da Silva, H.,
    2. L. K. Beura,
    3. H. Wang,
    4. E. A. Hanse,
    5. R. Gore,
    6. M. C. Scott,
    7. D. A. Walsh,
    8. K. E. Block,
    9. R. Fonseca,
    10. Y. Yan, et al
    . 2018. The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8+ T cells. Nature 559: 264–268.
    OpenUrl
  18. ↵
    1. Solle, M.,
    2. J. Labasi,
    3. D. G. Perregaux,
    4. E. Stam,
    5. N. Petrushova,
    6. B. H. Koller,
    7. R. J. Griffiths,
    8. C. A. Gabel
    . 2001. Altered cytokine production in mice lacking P2X(7) receptors. J. Biol. Chem. 276: 125–132.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Barnden, M. J.,
    2. J. Allison,
    3. W. R. Heath,
    4. F. R. Carbone
    . 1998. Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76: 34–40.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Zhong, J.,
    2. Z. Ye,
    3. S. W. Lenz,
    4. C. R. Clark,
    5. A. Bharucha,
    6. G. Farrugia,
    7. K. D. Robertson,
    8. Z. Zhang,
    9. T. Ordog,
    10. J. H. Lee
    . 2017. Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application. BMC Genomics 18: 985.
    OpenUrl
  21. ↵
    1. Pchelintsev, N. A.,
    2. P. D. Adams,
    3. D. M. Nelson
    . 2016. Critical parameters for efficient sonication and improved chromatin immunoprecipitation of high molecular weight proteins. PLoS One 11: e0148023.
    OpenUrlCrossRef
  22. ↵
    1. Mombaerts, P.,
    2. A. R. Clarke,
    3. M. A. Rudnicki,
    4. J. Iacomini,
    5. S. Itohara,
    6. J. J. Lafaille,
    7. L. Wang,
    8. Y. Ichikawa,
    9. R. Jaenisch,
    10. M. L. Hooper, et al
    . 1992. Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages. [Published erratum appears in 1992 Nature 360: 491.] Nature 360: 225–231.
    OpenUrlCrossRefPubMed
    1. Broussard, C.,
    2. C. Fleischacker,
    3. R. Horai,
    4. M. Chetana,
    5. A. M. Venegas,
    6. L. L. Sharp,
    7. S. M. Hedrick,
    8. B. J. Fowlkes,
    9. P. L. Schwartzberg
    . 2006. Altered development of CD8+ T cell lineages in mice deficient for the Tec kinases Itk and Rlk. [Published erratum appears in 2006 Immunity 25: 849.] Immunity 25: 93–104.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Palacios, E. H.,
    2. A. Weiss
    . 2007. Distinct roles for Syk and ZAP-70 during early thymocyte development. J. Exp. Med. 204: 1703–1715.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Ma, A.,
    2. J. C. Pena,
    3. B. Chang,
    4. E. Margosian,
    5. L. Davidson,
    6. F. W. Alt,
    7. C. B. Thompson
    . 1995. Bclx regulates the survival of double-positive thymocytes. Proc. Natl. Acad. Sci. USA 92: 4763–4767.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Freedman, B. D.,
    2. Q. H. Liu,
    3. G. Gaulton,
    4. M. I. Kotlikoff,
    5. J. Hescheler,
    6. B. K. Fleischmann
    . 1999. ATP-evoked Ca2+ transients and currents in murine thymocytes: possible role for P2X receptors in death by neglect. Eur. J. Immunol. 29: 1635–1646.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Szondy, Z.,
    2. É. Garabuczi,
    3. K. Tóth,
    4. B. Kiss,
    5. K. Köröskényi
    . 2012. Thymocyte death by neglect: contribution of engulfing macrophages. Eur. J. Immunol. 42: 1662–1667.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Surprenant, A.,
    2. F. Rassendren,
    3. E. Kawashima,
    4. R. A. North,
    5. G. Buell
    . 1996. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science 272: 735–738.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Cankurtaran-Sayar, S.,
    2. K. Sayar,
    3. M. Ugur
    . 2009. P2X7 receptor activates multiple selective dye-permeation pathways in RAW 264.7 and human embryonic kidney 293 cells. Mol. Pharmacol. 76: 1323–1332.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Zentner, G. E.,
    2. P. J. Tesar,
    3. P. C. Scacheri
    . 2011. Epigenetic signatures distinguish multiple classes of enhancers with distinct cellular functions. Genome Res. 21: 1273–1283.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Vanhille, L.,
    2. A. Griffon,
    3. M. A. Maqbool,
    4. J. Zacarias-Cabeza,
    5. L. T. Dao,
    6. N. Fernandez,
    7. B. Ballester,
    8. J. C. Andrau,
    9. S. Spicuglia
    . 2015. High-throughput and quantitative assessment of enhancer activity in mammals by CapStarr-seq. Nat. Commun. 6: 6905.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Hashimoto-Hill, S.,
    2. L. Friesen,
    3. M. Kim,
    4. C. H. Kim
    . 2017. Contraction of intestinal effector T cells by retinoic acid-induced purinergic receptor P2X7. Mucosal Immunol. 10: 912–923.
    OpenUrl
  32. ↵
    1. Fauber, B. P.,
    2. S. Magnuson
    . 2014. Modulators of the nuclear receptor retinoic acid receptor-related orphan receptor-γ (RORγ or RORc). J. Med. Chem. 57: 5871–5892.
    OpenUrl
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The Journal of Immunology: 202 (4)
The Journal of Immunology
Vol. 202, Issue 4
15 Feb 2019
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Cutting Edge: HDAC3 Protects Double-Positive Thymocytes from P2X7 Receptor–Induced Cell Death
Rachael L. Philips, Shaylene A. McCue, Matthew J. Rajcula, Virginia S. Shapiro
The Journal of Immunology February 15, 2019, 202 (4) 1033-1038; DOI: 10.4049/jimmunol.1801438

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Cutting Edge: HDAC3 Protects Double-Positive Thymocytes from P2X7 Receptor–Induced Cell Death
Rachael L. Philips, Shaylene A. McCue, Matthew J. Rajcula, Virginia S. Shapiro
The Journal of Immunology February 15, 2019, 202 (4) 1033-1038; DOI: 10.4049/jimmunol.1801438
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