Posttranslational modification, such as phosphorylation, is an important biological event that modulates and diversifies protein function. Bcl11b protein is a zinc-finger transcription factor that plays a crucial role in early T cell development and the segregation of T cell subsets. Bcl11b possesses at least 25 serine/threonine (S/T) residues that can be phosphorylated upon TCR stimulation. To understand the physiological relevance of the phosphorylation on Bcl11b protein, we replaced S/T residues with alanine (A) by targeting murine Bcl11b gene in embryonic stem cells. By combinational targeting of exons 2 and 4 in the Bcl11b gene, we generated a mouse strain, Bcl11b-phosphorylation site mutation mice, in which 23 S/T residues were replaced with A residues. Such extensive manipulation left only five putative phosphorylated residues, two of which were specific for mutant protein, and resulted in reduced amounts of Bcl11b protein. However, primary T cell development in the thymus, as well as the maintenance of peripheral T cells, remained intact even after loss of major physiological phosphorylation. In addition, in vitro differentiation of CD4+ naive T cells into effector Th cell subsets—Th1, Th2, Th17, and regulatory T—was comparable between wild-type and Bcl11b-phosphorylation site mutation mice. These findings indicate that the physiological phosphorylation on major 23 S/T residues in Bcl11b is dispensable for Bcl11b functions in early T cell development and effector Th cell differentiation.

Proteins often undergo posttranslational modifications (PTMs), such as phosphorylation, acetylation, methylation, SUMOylation, and ubiquitination. PTMs affect conformational changes, activation, molecular interactions, and stability of proteins. Thus, PTMs are one of the important biochemical reactions to diversify protein functions. Dysregulation of PTMs has been shown to cause several human diseases, including cancer. Therefore, a variety of molecules that possess catalytic activity for PTMs are candidate targets for cancer therapy (1, 2). Thus, it is important to understand how the function of each protein is modulated by PTMs under both physiological and pathological conditions. Some PTMs involve the function of two distinct types of enzyme, i.e., writers and erasers. For instance, the phosphorylation state of a protein is actively and negatively controlled by kinases and phosphatases, respectively. In cellular signaling pathways in lymphocytes, protein phosphorylation is induced by the stimulation of surface receptors, such as TCRs. TCR engagement by peptides present on MHC triggers a kinase cascade to activate T lymphocytes. The phosphorylation state is often associated with the active state of the cells (3).

The B cell leukemia/lymphoma 11B (Bcl11b) gene encodes a Krüppel-like transcription factor that possesses six C2H2-type zinc-finger (ZnF) domains (4, 5). Loss-of-function studies on Bcl11b gene in mice demonstrated that Bcl11b is essential for early T cell development (6–8). For instance, germline null mutation of Bcl11b impairs thymopoiesis and causes a complete developmental blockage during transition from double-negative 2 (DN2) to DN3 stage during early αβ T cell development (9). Using mass spectrometry with primary mouse thymocytes, Zhang et al. (10) reported that Bcl11b protein undergoes at least two types of PTM, phosphorylation of serine (S) and threonine (T) residues, and SUMOylation at lysine (K) residues, upon PMA/ionomycin stimulation, which mimics TCR stimulation. Twenty-five S/T residues of Bcl11b have been shown to undergo phosphorylation, and two residues (K679 and K877) accept SUMOylation. These observations suggest that the functional state of Bcl11b might be regulated by these PTMs. In particular, phosphorylation of multiple S/T residues raises the possibility that the phosphorylation state of Bcl11b, which could be dependent on the gradient of phosphorylated residues, might not only serve as a crucial regulator of Bcl11b function but also as a transmitter of TCR signal strength to a transcriptional program that governs thymocyte fate determination (11, 12).

In this study, we have used genetic approaches in mice to examine whether phosphorylation of the S/T residues of Bcl11b plays a crucial role in regulating its functions. We show that primary T cell development in the thymus and αβT cells in the peripheral lymphocyte pool are not affected by the replacement of 23 S/T residues of Bcl11b protein with alanine (A). Without phosphorylation of the earlier 23 S/T residues, CD4+ T cells are differentiated into several effector Th cell subsets in in vitro cultures. These findings indicated that major physiological phosphorylation of 23 S/T residues in Bcl11b is dispensable for the major function of Bcl11b to support early T cell development, maintain the peripheral T cell pool, and ensure differentiation into several effector CD4+ T cell subsets in vitro and type 2 immune responses against Alternaria alternata Ags in vivo.

To construct a vector that would target A replacement mutation onto four S residues encoded by exon 2 of murine Bcl11b gene, we obtained the target vector (a gift from Dr. Katsuragi) that was used to generate Bcl11bEX2flox mice (13). To create a mutant DNA fragment harboring corresponding four S/T with A replacement mutation, we performed overlap PCR, followed by confirmation of sequences. We then replaced the region containing exon 2 in the original targeting vector with mutant exon 2 fragment by DNA ligation. During this process, loxP sequences downstream of the neor (neomycin-resistant gene) gene cassette were removed and shortened the 5′ homology arm to enable us to set up PCR screening for homologous recombination, generating the targeting vector version 1 that harbors a 1040-bp-length 5′ homology region.

For the gene targeting combined with a CRISPR/Cas9-mediated double-strand break, we searched candidate target sequences for single guide RNA (gRNA) by the Broad Institute CRISPick Web site (https://portals.broadinstitute.org/gppx/crispick/public) and selected 5′-AGCAGAGGCTGACCATGTGG-AGG(PAM)-3′. Synthesized sense- and antisense-oligonucleotides were annealed and ligated into the BbsI site in the pX330 vector (#42230; Addgene), generating pX330-Bcl11bEX2 vector. To avoid sequential double-strand break by CRISPR/Cas9 in the target allele, we mutated a sequence corresponding to gRNA target to 5′-AGCAGAGGCTGACCATGTCG-AGG-3′ by site-specific PCR-based mutagenesis, generating the targeting vector version 2. A total of 5 μg of pX330-Bcl11bEX2 vector and 15 μg of the nonlinearized targeting vector version 2 were transfected into the M1 embryonic stem (ES) cell line by using Xfect mESC transfection reagent (Clontech 631320; Clontech Laboratories, CA) according to the manufacturer’s protocol. Screening of ES clones that underwent homologous recombination was performed by PCR. After isolation of 14 candidate ES clones, we examined sequences around exon 2 and selected one clone that harbors A replacement at three S residues (S95, S96, and S109), which is hereafter referred to as Bcl11bE2PM (the exon 2 phosphorylation site mutation [PM]) mutation in this article. Removal of the neor gene flanked with FRT sites was performed by transient transfection of an expression vector encoding Flp recombinase.

To construct the targeting vector that would replace 20 S/T residues encoded by exon 4 of murine Bcl11b gene with A, which is hereafter referred to as Bcl11bE4PM mutation, we ordered custom dsDNA synthesis service (Eurofins Genomics, Luxembourg, Luxembourg). Synthesized DNA fragment was used to replace the part of the exon 4 sequence in the target vector that was used to generate the Bcl11bEX4flox allele (14). A total of 30 μg of linearized targeting vector was transfected into M1 ES cells by electroporation, and screening of ES clones that underwent homologous recombination was performed as previously described (14).

To combine the Bcl11bE2PM and Bcl11bE4PM mutations, we transfected the targeting vector for the Bcl11bE4PM mutation into the ES clone harboring Bcl11b+/E2PM genotype. After isolation of ES clones that underwent homologous recombination with the targeting vector, we examined which allele, Bcl11b or Bcl11bE2PM, underwent homologous recombination by transduction of candidate ES clones with Cre retroviral vector. Because the direction of loxP sequence at upstream of exon 2 and downstream of exon 4 is opposite, detection of reverse type of site-specific recombination by PCR was evaluated as a signature for targeting the Bcl11bE4PM mutation onto the Bcl11bE2PM allele. From such candidate ES clone, we remove the neor gene flanked with loxP site by transient transfection of the Cre expression vector. G418-sensitive ES clones were isolated, and their genotypes were confirmed by several types of genomic PCR.

Generation of chimera mice from M1 ES cells by aggregation was performed by the RIKEN center for integrative medical sciences animal facility as previously described (14). All the animals were bred and maintained under the specific pathogen-free condition and were handled in accordance with institutional guidelines for animal care and with a protocol approved by Institutional Animal Care and Use Committee of RIKEN Yokohama Branch.

Total thymocytes were isolated from mice by mashing the thymus with the plunger of a 2.5-ml syringe and a 40-μm pore cell strainer (BD Biosciences, Franklin Lakes, NJ). Freshly isolated thymocytes were lysed with RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Waltham, MA) containing Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific) by incubating on ice for 10 min, and cell debris was removed by centrifugation at 15,000 × g for 10 min. To dephosphorylate the samples, we treated an aliquot of lysate with alkaline phosphatase (AP; Roche, Basel, Switzerland) for 1 h at 37°C, followed by incubating for 10 min at 55°C to inactivate the enzyme activity. Lysate was mixed with 1× volume of 2× Laemmli Sample Buffer (Bio-Rad, Hercules, CA) and boiled at 95°C for 10 min. Electrophoresis was performed with 10% polyacrylamide gel (e-PAGEL; ATTO, Tokyo, Japan), and proteins were transferred onto polyvinylidene difluoride membrane (Merck Millipore, Burlington, MA). After blocking with 1× TBS-T buffer (nacalai tesque, Kyoto, Japan) with 5% (w/v) skim milk (Wako, Osaka, Japan), we split the membrane into two pieces at appropriate position and incubated with following primary Abs in 1× TBS-T overnight at 4°C: upper piece with rabbit anti-mouse Bcl11b polyclonal IgG (A300-383A; Bethyl Laboratories, Montgomery, TX) or rabbit anti-SUMO-1 polyclonal IgG (#4930; Cell Signaling Technology, Danvers, MA), and lower piece with rabbit anti-human/mouse G3PDH/Gapdh polyclonal IgG (#2275-PC-100; R&D Systems, Minneapolis, MN). Membrane was washed with 1× TBS-T several times and incubated with HRP-conjugated donkey anti-rabbit IgG polyclonal IgG (NA934; GE Healthcare, Chicago, IL) for 30 min at room temperature. Target proteins were developed by use of Amersham ECL Select Western blotting Detection Reagent (GE Healthcare) and Amersham Imager 680 (GE Healthcare). Densitometry was done with ImageJ software.

Total RNAs were extracted from thymocytes by use of RNeasy Mini Kit (Qiagen, Hilden, Germany) and RNase-Free DNase Set (Qiagen) according to the manufacturer’s protocol. Concentrations of isolated RNAs were measured with NanoDrop One (Thermo Fisher Scientific), and 100 ng of RNAs was reverse transcribed with SuperScript IV First-Strand Synthesis System and the oligo(dT)20 primer (Thermo Fisher Scientific). Expressions of Bcl11b (NM_001079883.1) and Hprt (NM_013556.2) were measured by use of PowerUp SYBR Green Master Mix (Thermo Fisher Scientific), specific primers (Eurofins Genomics), and QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). Designed primers are as follows: Bcl11b forward: 5′-TGTCCCAGAGGGAACTCATC-3′, Bcl11b reverse: 5′-CAGGCTGCTAGGCTCCTCTA-3′; and Hprt forward: 5′-GTCGTGATTAGCGATGATGAACC-3′, Hprt reverse: 5′-ATGACATCTCGAGCAAGTCTTTCAG-3′.

Total thymocytes were isolated from mice by mashing the thymus. One million cells were lysed in 1× Laemmli Sample Buffer containing 2-ME by boiling at 95°C for 10 min, and the lysate was stored as untreated input (0 h). Remaining cells were suspended to customized DMEM (containing penicillin-streptomycin, 2-ME, sodium pyruvate, and HEPES) (Kohjin Bio, Saitama, Japan) supplemented with 10% (v/v) FBS (GE Healthcare) at a density of 106 cells/ml. One milliliter of suspension was plated on the wells of a 24-well plate and treated with 200 μg/ml cycloheximide (CHX; nacalai tesque) for 4 h. After the incubation, cells were harvested, washed with ice-cold D-PBS(−) (nacalai tesque) several times, and lysed in 1× Laemmli Sample Buffer by boiling. The amount of Bcl11b in 105 cells was measured by immunoblotting. Half-life (t1/2) of Bcl11b protein was calculated with the following formula: N4=N0 ×(12)4t1/2, where N4 is the amount of Bcl11b after the 4-h CHX treatment and N0 is the amount of Bcl11b in input (0 h). MG-132 was purchased from Merck Millipore and reconstituted in DMSO. Freshly isolated thymocytes were incubated for 6 h in the presence of 20 μM MG-132. As control, cells were treated with DMSO. Cells were harvested after the incubation and lysed in 1× Laemmli Sample Buffer.

CD4 T cells were isolated from OT-II mouse spleen by use of MojoSort Mouse CD4 T cells Isolation kit (BioLegend, San Diego, CA). OT-II CD4 T cells (5 × 105 cells) and irradiated splenic cells from C57BL/6N mice (2.5 × 106 cells) were cultured with chicken OVA 323–339 (1 μM). In vitro skewing was performed by the addition of anti–IL-4 mAb (1 μg/ml, clone: 11B11; PeproTech, Cranbury, NJ) and IL-12 (10 ng/ml; PeproTech) for Th1; rmIL-4 (10 ng/ml; PeproTech) and anti–IFN-γ mAb (1 μg/ml, clone: R4-6A2; BD Biosciences) for Th2; IL-6 (20 ng/ml; PeproTech), TGF-β (5 ng/ml; R&D Systems), anti–IL-4, and anti–IFN-γ mAbs for Th17; and TGF-β, anti–IL-4, and anti–IFN-γ mAbs for regulatory T (Treg). After 6 d, cells were restimulated with anti-TCRβ mAb for 6 h in the presence of 2 μM monensin (Sigma, St. Louis, MO). The cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. After blocking with 3% BSA-PBS, mouse cells were stained with the following mAbs: IL-4 (clone: 11B11; BD Biosciences), IFN-γ (clone: XNG1.2; BD Biosciences), IL-17 (clone: TC11-18H10; BD Biosciences), and Foxp3 (clone: FJK-16s; eBioscience).

Total thymocytes or splenocytes were isolated by mashing the organs with the plunger of a 2.5-ml syringe on a 40-μm pore cell strainer. Liver was chopped with scissors, mashed on a cell strainer, and suspended to RPMI-1640 medium (nacalai tesque) with 5% (v/v) FBS. Percoll was added at a final concentration of 14% (v/v), and cells were pelleted by centrifugation. RBCs were lysed with ACK lysis buffer (Thermo Fisher Scientific). Intraepithelial lymphocytes (IELs) were isolated from the small intestine. After the removal of feces and Peyer’s patches, the small intestine was incubated in RPMI-1640 medium supplemented with 2% (v/v) FBS and 5 mM EDTA at 37°C for 20 min with gentle rotating (200 rpm). After vigorous vortex, floating cells were harvested as IELs. IELs were further purified with a 40 and 80% (v/v) Percoll gradient. Cells were washed with ice-cold D-PBS(−) supplemented with 5% FBS and 0.09% NaN3 (Wako). After Fc receptor blocking with 0.5 μg/106 cells of 2.4G2 Ab (BD Biosciences), cells were stained with Abs for surface markers on ice for 30 min. Cells were washed twice, resuspended in D-PBS(−) supplemented with 1.25 μg/ml 7-aminoactinomycin D (BD Biosciences) to distinguish dead cells, and analyzed with BD FACSCanto II (BD Biosciences). Data were analyzed with FlowJo software (BD Biosciences). Abs used in this experiment were purchased from BD Biosciences or eBioscience: CD4 (clone: RM4-5), CD8α (clone: 53-6.7), CD8β (clone: H35-17.2), CD24 (clone: M1/69), TCRβ (clone: H57-597), TCRγδ (clone: GL3), CD3ε (clone: 145-2C11), CD44 (clone: IM7), CD25 (clone: PC61.5), CD122 (clone: TM-b1), and CD62L (clone: MEL-14). Α-galactosyl ceramide (αGalCer)-loaded CD1d tetramer was prepared by mixing recombinant dimeric mouse CD1d:Ig fusion protein (DimerX I; BD Biosciences), αGalCer, and anti-mouse IgG1 Ab.

Thymocytes or splenic T cells were analyzed with following gating strategies (Supplemental Fig. 1). Thymocytes or lymphocytes were first gated based on forward and side scatter area, and doublets were excluded as forward scatter wide high population. Live cells were determined as 7-aminoactinomycin D–negative cells. DN1–4 thymocytes were determined by CD44 and CD25 expressions on CD4CD8α thymocytes. Mature thymocytes were defined as CD24low TCRβ+ cells, and splenic T cells were identified as CD3e+TCRβ+ cells. Nonconventional γδT cells, invariant NKT (iNKT) cells, and IEL subsets were analyzed with the following gating strategies (Supplemental Fig. 4). Live singlets were first gated. In the thymus and spleen, γδT cells were defined as TCRγδ+ cells in CD24 and CD3ε+ cells, respectively. Liver and IEL γδT cells were identified as CD45+TCRγδ+ cells. iNKT cells were investigated in the liver using αGalCer-loaded CD1d tetramer in CD45+TCRβ+NK1.1+ cells. In IELs, cytotoxic CD4 and CD8αα T cells were examined as CD4+CD8α+ and CD8α+CD8β cells in CD45+TCRβ+ cells, respectively.

Total thymocytes were isolated from mice by mashing the thymus with the plunger of a 2.5-ml syringe and a 70-μm pore cell strainer. Freshly isolated 2 × 107 thymocytes were suspended to DMEM supplemented with 10% (v/v) FBS and antibiotics and incubated at 37°C. After 4 h of incubation, cells were stimulated with 100 ng/ml PMA and 500 ng/ml ionomycin for 30 min. Cells were harvested, washed with ice-cold D-PBS(−) twice, and lysed in RIPA Lysis and Extraction Buffer supplemented with Halt Protease Inhibitor Cocktail, Halt Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), 5 mM N-ethylmaleimide, and 2 U/ml Benzonase (Sigma) by incubating for 30 min at 4°C with gentle rotation. After removal of cell debris by the centrifugation at maximum speed, cell lysate was mixed with 5 μg of rabbit anti-mouse Bcl11b Ab (A300-385A; Bethyl Laboratories) prebound to 50 μl of Dynabeads Protein G (Thermo Fisher Scientific) and incubated overnight at 4°C with gentle rotation. Protein-beads complex was washed with 50 mM ammonium bicarbonate several times and resuspended to 50 μl of 50 mM ammonium bicarbonate.

Proteins on the beads were digested with 400 ng trypsin/LysC mix (Promega, WI) at 37°C overnight. The digests were reduced, alkylated, acidified, and desalted using GL-Tip SDB (GL Sciences, Tokyo, Japan). The eluates were evaporated and dissolved in 3% acetonitrile (ACN) and 0.1% trifluoroacetic acid. Liquid chromatography (LC)-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a 75-μm inner diameter × 150 mm C18 reversed-phase column (Nikkyo Technos, Tokyo, Japan) with a linear 4–32% ACN gradient for 0–100 min, followed by an increase to 80% ACN for 10 min and finally held at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with the top 10 MS/MS methods. MS1 spectra were measured with a resolution of 70,000, an automatic gain control target of 1e6, and a mass range from 350 to 1500 m/z. Higher energy collisional dissociation MS/MS spectra were acquired at a resolution of 17,500, an automatic gain control target of 5e4, an isolation window of 2.0 m/z, a maximum injection time of 60 ms, and a normalized collision energy of 27. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the SwissProt database restricted to Mus musculus supplemented with the Bcl11bE2*4PM sequence using Proteome Discoverer v2.5 (Thermo Fisher Scientific) with the Sequest HT search engine. The search parameters were as follows: (1) trypsin as an enzyme with up to two missed cleavages; (2) precursor mass tolerance of 10 ppm; (3) fragment mass tolerance of 0.02 Da; (4) N-ethylmaleimide modification of cysteine as a fixed modification; and (5) acetylation of the protein N terminus, oxidation of methionine, di-glycine modification of lysine, and phosphorylation of S, T, and tyrosine (Y) as variable modifications. Peptides were filtered at a false discovery rate of 1% using the Percolator node.

Female mice were anesthetized by isoflurane inhalation, followed by intranasal injection of A. alternata extract [10117, 20 μg/head, in 40 μl of D-PBS(−); ITEA, Tokyo, Japan] on days 0, 3, and 6. At 24 h after final challenge, naive and A. alternata–treated female mice were euthanized by CO2 inhalation. For cytokine analyses, the bronchial alveolar lavage fluid (BALF) was first collected by intratracheal insertion of a catheter and one lavage of 500 μl of HBSS (nacalai tesque) containing 2% (v/v) FBS and transferred into new 1.5-ml Eppendorf tubes. This procedure was further repeated twice, totaling to a final volume of 1.5 ml. BALF cells were kept at 4°C for flow-cytometric analyses. For the preparation of lung cell suspensions, the lungs were first perfused with 20 ml of D-PBS(-) through the left ventricle of the heart. Perfused lungs were dissected out and minced up before incubation in RPMI-1640 supplemented with 2% (v/v) FBS, 0.3 mg/ml collagenase IV (Sigma), and 0.3 mg/ml DNase I (Wako) at 37°C for 45 min with continuous shaking. To homogenize the samples, we mashed the digested lung tissues in a 70-μm cell strainer in a petri dish, and cell suspensions were then pelleted by centrifugation at 300 × g at 4°C for 5 min. For the elimination of debris, the cell pellets were resuspended in 10 ml RPMI-1640 medium containing 2% (v/v) FBS and 30% (v/v) Percoll and separated by centrifugation at 860 × g at room temperature for 20 min with brakes off. Debris in the upper phase was aspirated off, and cell pellets were resuspended in 5 ml RPMI-1640 with 2% (v/v) FBS. Cells were washed and pelleted by centrifugation at 300 × g at 4°C for 5 min and were then subjected to analysis. For ex vivo stimulation, Th2 cells (CD45+Thy1.2+CD4+ST2+) were sorted using FACSAria. Twenty thousand Th2 cells were activated in 100 ng/ml PMA and 500 ng/ml ionomycin in a round-bottom 96-well plate for 4 h, in the presence of 2 μM monensin, and were then subjected to flow-cytometric analyses for intracellular cytokines levels. Abs used in this experiment were purchased from BD Biosciences or eBioscience: CD4 (clone: RM4-5), CD11c (clone: HL3), CD45 (clone: 30-F11), Gata3 (clone: L50-823), IL-4 (clone: 11B11), IL-5 (clone: TRFK5), Siglec-F (clone: E13-161.7), ST2 (clone: U29-93), and TCRβ (clone: H57-597).

Bcl11b protein was shown to possess 25 S/T residues that are phosphorylated in primary mouse thymocytes in response to PMA/ionomycin stimulation (10). Among these residues, four residues (S95, S96, S109, and S128) near the N terminus are encoded by exon 2, whereas 20 residues (T260, S277, T313, S318, T376, S381, S398, S401, T406, T416, S496, S664, S731, S734, T744, S762, S765, T766, S778, and S779) are encoded by exon 4 (Supplemental Fig. 2). To investigate the biological significance of phosphorylation of the Bcl11b protein, we designed target vectors to replace S/T residues, encoded by either exon 2 or exon 4, with A residues. Unfortunately, despite screening 472 G418-resistant ES clones, we were unable to isolate ES clones that underwent homologous recombination with the targeting vector version 1 for exon 2 by conventional gene-targeting strategy. To overcome this problem, we tested whether targeting DNA double-strand breaks by CRISPR/Cas9 would increase the efficiency of homologous recombination (Fig. 1A). A combination of CRISPR/Cas9-mediated double-strand breaks and transfection of the circular targeting vector version 2 resulted in a dramatic increase in the efficiency of homologous recombination. This resulted in homologous recombination in 5 of 96 G418-resistant ES clones. However, we noticed that these clones often contained insertion/deletion (indel) type mutations near the putative double-strand break point. After sequencing 14 candidate ES clones, we obtained one ES clone in which three S residues (S95, S96, and S109), located near the double-strand break point, were replaced with A, whereas the S128 residue remained unchanged. At this stage, we decided to use this ES clone to generate a mouse line, instead of repeating transfection to obtain ES clones with all four S residues replaced with A. Hereafter, we refer to this S-to-A replacement mutation on S95, S96, and S109 residues on Bcl11b protein as the Bcl11bE2PM (the exon 2 phosphorylation site mutation [PM]) mutation.

FIGURE 1.

Generation of Bcl11b-PM mutant mouse lines. (A) A scheme of targeting mutations onto exon 2 in murine Bcl11b gene. CRISPR/Cas9-mediated DNA double-strand break was combined with conventional gene targeting. Structure of Bcl11b gene, targeting vector, and targeted Bcl11b allele. Arrowhead, black box; red triangle and green ovals indicate the position of single gRNA, exon 2, loxP site, and FRT sites, respectively. Sequences data showing mutations introduced to replace S95, S96, and S109 to A. (B) Sequences around the Bcl11bE4PM allele. DNA sequences of coding region are shown as capital letters. Mutations to replace S/T residues to A are shown in red font. (C) Strategy for targeting 10 Bcl11bE4PM mutations. Structure of wt Bcl11b and mutant Bcl11bE2PM alleles, targeting vector, and targeted Bcl11b allele. Black box, gray box, red triangle, and arrows indicate wt exons, mutated exons, and loxP site and positions of PCR primers to detect Cre-mediated reverse-type recombination, respectively. Gel image showing the result of PCR screening for ES clones underwent homologous recombination with the targeting vector.

FIGURE 1.

Generation of Bcl11b-PM mutant mouse lines. (A) A scheme of targeting mutations onto exon 2 in murine Bcl11b gene. CRISPR/Cas9-mediated DNA double-strand break was combined with conventional gene targeting. Structure of Bcl11b gene, targeting vector, and targeted Bcl11b allele. Arrowhead, black box; red triangle and green ovals indicate the position of single gRNA, exon 2, loxP site, and FRT sites, respectively. Sequences data showing mutations introduced to replace S95, S96, and S109 to A. (B) Sequences around the Bcl11bE4PM allele. DNA sequences of coding region are shown as capital letters. Mutations to replace S/T residues to A are shown in red font. (C) Strategy for targeting 10 Bcl11bE4PM mutations. Structure of wt Bcl11b and mutant Bcl11bE2PM alleles, targeting vector, and targeted Bcl11b allele. Black box, gray box, red triangle, and arrows indicate wt exons, mutated exons, and loxP site and positions of PCR primers to detect Cre-mediated reverse-type recombination, respectively. Gel image showing the result of PCR screening for ES clones underwent homologous recombination with the targeting vector.

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Similarly, we designed mutations that replaced the 20 S/T residues encoded by exon 4 (Fig. 1B), which is referred to as the Bcl11bE4PM mutation, and synthesized a dsDNA fragment to construct a plasmid to target the Bcl11bE4PM mutation into the Bcl11b gene. In contrast with that of Bcl11bE2PM targeting, the efficiency of Bcl11bE4PM targeting in ES cells was higher, and 1 of 32 G418-resistant ES clones underwent homologous recombination with the targeting vector. Encouraged by these results, we attempted to combine the Bcl11bE2PM mutation with the Bcl11bE4PM mutation, which we referred as the Bcl11bE2*4PM mutation. This mutation harbors A replacement at 23 S/T residues; this was obtained by transfection of the ES clone with the Bcl11b+/E2PM genotype with the Bcl11bE4PM targeting vector. To identify ES clones that exhibited homologous recombination with the Bcl11bE4PM targeting vector in the Bcl11bE2PM allele, and not in the wild-type (wt) Bcl11b allele, we examined a Cre-mediated reverse type of recombination between a loxP site upstream of exon 2 and downstream of coding region in exon 4 after retroviral Cre transduction (Fig. 1C). After removing the neor gene by transient transfection of the Cre expression plasmid, ES clones with appropriate genotypes were used to generate chimeric mice, and using them, we established Bcl11bE2PM, Bcl11bE4PM, and Bcl11bE2*4PM mutant mouse lines (Fig. 2A). LC-MS/MS analysis of Bcl11b protein immunoprecipitated from the Bcl11bE2*4PM/E2*4PM mouse thymocytes validated the successful replacement of 23 S/T phosphorylation-accepting residues with A in Bcl11bE2*4PM mutant protein (Supplemental Fig. 2B).

FIGURE 2.

Growth retardation of Bcl11bE4PM/E4PM and Bcl11bE2*4PM/E2*4PM mice. (A) Scheme of Bcl11b gene (ENSMUSG00000048251) and a map of 24 S/T residues (black lines) that can be phosphorylated within a Bcl11b protein. Exons 2, 3, and 4 encode 4, 1, and 20 S/T residues, respectively. Introduced A replacements were shown as red lines. (B) Body weights of Bcl11b+/+ (n = 8, cohoused), Bcl11b+/Δ (n = 6, cohoused), Bcl11bE2PM/E2PM (n = 8, cohoused), Bcl11bE4PM/E4PM (n = 3, cohoused), and Bcl11bE2*4PM/E2*4PM (n = 7, cohoused) mice at 21 days old are shown at left. Body weights of Bcl11b+/+ (n = 4, littermates), Bcl11b+/E2*4PM (n = 9, littermates), and Bcl11bE2*4PM/E2*4PM (n = 3, littermates) mice at 7 weeks old are shown at right. (C) Growth curve of Bcl11b+/+ (n = 9), Bcl11b+/E2*4PM (n = 9), and Bcl11bE2*4PM/E2*4PM (n = 4) littermates from 2 to 4 weeks after birth. Bcl11bE2*4PM/E2*4PM mice show the growth retardation. Error bar indicates 95% CI. ***p < 0.0005.

FIGURE 2.

Growth retardation of Bcl11bE4PM/E4PM and Bcl11bE2*4PM/E2*4PM mice. (A) Scheme of Bcl11b gene (ENSMUSG00000048251) and a map of 24 S/T residues (black lines) that can be phosphorylated within a Bcl11b protein. Exons 2, 3, and 4 encode 4, 1, and 20 S/T residues, respectively. Introduced A replacements were shown as red lines. (B) Body weights of Bcl11b+/+ (n = 8, cohoused), Bcl11b+/Δ (n = 6, cohoused), Bcl11bE2PM/E2PM (n = 8, cohoused), Bcl11bE4PM/E4PM (n = 3, cohoused), and Bcl11bE2*4PM/E2*4PM (n = 7, cohoused) mice at 21 days old are shown at left. Body weights of Bcl11b+/+ (n = 4, littermates), Bcl11b+/E2*4PM (n = 9, littermates), and Bcl11bE2*4PM/E2*4PM (n = 3, littermates) mice at 7 weeks old are shown at right. (C) Growth curve of Bcl11b+/+ (n = 9), Bcl11b+/E2*4PM (n = 9), and Bcl11bE2*4PM/E2*4PM (n = 4) littermates from 2 to 4 weeks after birth. Bcl11bE2*4PM/E2*4PM mice show the growth retardation. Error bar indicates 95% CI. ***p < 0.0005.

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Bcl11b deficiency caused by germline Bcl11b null mutation leads to neonatal lethality, and Bcl11bΔ/Δ mice die within a few days after birth (15). In contrast, homozygous mice with either Bcl11bE2PM, Bcl11bE4PM, or Bcl11bE2*4PM mutation survived beyond the neonatal stage. However, Bcl11bE4PM/E4PM and Bcl11bE2*4PM/E2*4PM mice displayed growth retardation, and their body weight was lower than that of control wt mice at 3 wk of age (Fig. 2B). The body weight of the Bcl11bE2PM/E2PM mice was comparable with that of control littermates. In our animal facility, heterozygous mice with Bcl11b null mutation (Bcl11b+/Δ) also showed growth retardation to an extent similar to that observed in Bcl11bE4PM/E4PM and Bcl11bE2*4PM/E2*4PM mice at 3 wk of age (Fig. 2B) (16). We monitored the body weight of these mice from 2 to 4 wk of age and found that growth retardation was already apparent at 2 wk and lasted up to 4 wk of age (Fig. 2C). At 7 wk of age, the body weight of Bcl11bE2*4PM/E2*4PM mice became comparable with that of control Bcl11b+/+ and Bcl11b+/E2*4PM littermates (Fig. 2B), indicating that the growth retardation of mutant mice was specifically observed at young ages.

Given the growth retardation observed in Bcl11bE4PM/E4PM and Bcl11bE2*4PM/E2*4PM mice, as well as Bcl11b+/Δ mice, we next examined the protein levels of Bcl11b in total thymocytes by using immunoblotting with Abs that specifically recognize the N-terminal portion of Bcl11b (14). These Abs detected multiple Bcl11b bands. In addition to two major bands (indicated by the dagger symbol [†] in Fig. 3A), some faint signals were observed above these bands (indicated by paragraph symbol [¶] in Fig. 3A). The immunoblotting with anti-SUMO1 Ab after Bcl11b immunoprecipitation revealed that these bands corresponded to SUMOylated Bcl11b as previously reported (Fig. 3B) (17). The proportion of SUMOylated Bcl11b was higher in Bcl11bE4PM/E4PM and Bcl11bE2*4PM/E2*4PM mice than in the wt controls (Fig. 3B). Predicted molecular weights based on the amino acid sequences of wt and PD mutant Bcl11b proteins were as follows: wt Bcl11b (94.58 kDa), Bcl11bE2PM (94.53 kDa), Bc11bE4PM (94.16 kDa), and Bcl11bE2*4PM (94.11 kDa) (https://www.bioinformatics.org/sms/prot_mw.html) (18). Immunoblotting revealed accelerated mobilities of Bcl11bE4PM and Bcl11bE2*4PM proteins, indicating that the relative molecular masses of these proteins were lower than those of the wt Bcl11b and Bcl11bE2PM proteins (Fig. 3A, 3C). After treatment with AP, the relative molecular mass of wt and Bcl11bE2PM proteins as assessed by immunoblotting became reduced, suggesting that some residues of these Bcl11b proteins are phosphorylated in thymocytes even in the absence of PMA/ionomycin stimulation (Fig. 3A, Supplemental Fig. 3A). In contrast, AP treatment did not have any remarkable effect on the mobilities of Bcl11bE4PM and Bcl11bE2*4PM proteins (Supplemental Fig. 3A). These data indicated that at least some of the 20 S/T residues encoded by exon 4 served as the major phosphorylation sites for Bcl11b protein in primary thymocytes at steady state.

FIGURE 3.

Reduction of Bcl11b protein amount by the loss of major phosphorylation sites by the Bcl11bE2*4PM mutation. (A) Amount of Bcl11b proteins in thymocytes of Bcl11b+/+, Bcl11bE2PM/E2PM, Bcl11bE4PM/E4PM, and Bcl11bE2*4PM/E2*4PM mice were evaluated by immunoblotting. Cell lysates were treated with AP to dephosphorylate Bcl11b proteins. Gapdh was employed as the loading control. Representative Bcl11b and Gapdh immunoblotting images out of four experiments are shown. Relative quantities (RQs) to Bcl11b+/+ cells were shown. Un-SUMOylated Bcl11b; SUMOylated Bcl11b. (B) The SUMOylation in wt and E2*4PM mutant Bcl11b proteins was evaluated by immunoblotting with anti-SUMO1 antibody after Bcl11b immunoprecipitation. Proportions of SUMOylated Bcl11b in indicated genotypes are shown as average value ± 95% CI of at least four replicates. (C) Mobilities of wt and PD mutant Bcl11b proteins analyzed by densitometry are shown as histograms with protein size marker. (D) The phosphorylation in wt and E2*4PM Bcl11b mutant proteins was analyzed by LC-MS/MS. Phosphorylation states of reported 25 S/T and newly identified nine residues are shown. Color key indicates the probability of the phosphorylation. (E) RQs of Bcl11b proteins from indicated genotype cells (Bcl11b+/Δ [n = 3], Bcl11bE2PM/E2PM [n = 4], Bcl11bE4PM/E4PM [n = 6], and Bcl11bE2*4PM/E2*4PM [n = 5]) to Bcl11b+/+ cells (n = 10) are shown as average value ± 95% CI. (F) Expressions of Bcl11b mRNA in total thymocytes from indicated genotypes were determined by quantitative PCR. Mean (±95% CI) expressions relative to Hprt transcript are shown (Bcl11b+/+ [n = 4], Bcl11bE2PM/E2PM [n = 3], Bcl11bE4PM/E4PM [n = 3], and Bcl11bE2*4PM/E2*4PM [n = 3]). **p < 0.005, ***p < 0.0005.

FIGURE 3.

Reduction of Bcl11b protein amount by the loss of major phosphorylation sites by the Bcl11bE2*4PM mutation. (A) Amount of Bcl11b proteins in thymocytes of Bcl11b+/+, Bcl11bE2PM/E2PM, Bcl11bE4PM/E4PM, and Bcl11bE2*4PM/E2*4PM mice were evaluated by immunoblotting. Cell lysates were treated with AP to dephosphorylate Bcl11b proteins. Gapdh was employed as the loading control. Representative Bcl11b and Gapdh immunoblotting images out of four experiments are shown. Relative quantities (RQs) to Bcl11b+/+ cells were shown. Un-SUMOylated Bcl11b; SUMOylated Bcl11b. (B) The SUMOylation in wt and E2*4PM mutant Bcl11b proteins was evaluated by immunoblotting with anti-SUMO1 antibody after Bcl11b immunoprecipitation. Proportions of SUMOylated Bcl11b in indicated genotypes are shown as average value ± 95% CI of at least four replicates. (C) Mobilities of wt and PD mutant Bcl11b proteins analyzed by densitometry are shown as histograms with protein size marker. (D) The phosphorylation in wt and E2*4PM Bcl11b mutant proteins was analyzed by LC-MS/MS. Phosphorylation states of reported 25 S/T and newly identified nine residues are shown. Color key indicates the probability of the phosphorylation. (E) RQs of Bcl11b proteins from indicated genotype cells (Bcl11b+/Δ [n = 3], Bcl11bE2PM/E2PM [n = 4], Bcl11bE4PM/E4PM [n = 6], and Bcl11bE2*4PM/E2*4PM [n = 5]) to Bcl11b+/+ cells (n = 10) are shown as average value ± 95% CI. (F) Expressions of Bcl11b mRNA in total thymocytes from indicated genotypes were determined by quantitative PCR. Mean (±95% CI) expressions relative to Hprt transcript are shown (Bcl11b+/+ [n = 4], Bcl11bE2PM/E2PM [n = 3], Bcl11bE4PM/E4PM [n = 3], and Bcl11bE2*4PM/E2*4PM [n = 3]). **p < 0.005, ***p < 0.0005.

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To further investigate the phosphorylation in Bcl11b protein, we performed LC-MS/MS analysis. Total thymocytes from Bcl11b+/+ and Bcl11bE2*4PM/E2*4PM mice were incubated in the presence or absence of PMA/ionomycin, and Bcl11b proteins were purified by immunoprecipitation. LC-MS/MS analysis of the wt Bcl11b protein demonstrated the phosphorylation of S/T residues both in unstimulated and in stimulated conditions. We detected the phosphorylation on 21 of 25 reported residues, and we further identified, to our knowledge, seven novel putative phosphorylation acceptors (T130, S258, S374, S375, S405, S758, and T760) (Fig. 3D, Supplemental Fig. 2B). In addition, some of the phosphorylated residues appeared to be increased by PMA/ionomycin stimulation (Supplemental Fig. 3B). Analyses of Bcl11bE2*4PM protein, however, showed that replacement of S/T residues with A residues disrupted the phosphorylation of 23 S/T residues. As expected, S128, which we failed to manipulate on exon 2, and S169 encoded by exon 3 were phosphorylated, as well as wt protein (Fig. 3D, Supplemental Fig. 2B). To our knowledge, we found a novel phosphorylation site, T130, that is phosphorylated in both wt and Bcl11bE2*4PM protein. Interestingly, S487 and S725 residues were phosphorylated specifically in Bcl11bE2*4PM protein (Fig. 3D). This finding suggests that our extensive manipulation may have possibly converted these residues into de novo phosphorylation-accepting sites through yet uncharacterized mechanisms. Lastly, analyses of both wt and Bcl11bE2*4PM proteins revealed no phosphorylated Y residue (Supplemental Fig. 2B). Overall, the PM mutation disrupted the major physiological phosphorylation in Bcl11b protein, but Bcl11bE2*4PM mutant protein was still phosphorylated on at least five S/T residues, two of which were noncanonical cryptic phosphorylation sites.

The analysis of protein amounts using densitometry of the immunoblots showed that the amounts of Bcl11bE4PM and Bcl11bE2*4PM mutant proteins were significantly lower than those of wt Bcl11b protein and were estimated as 39.9 ± 10.6% (mean ± 95% confidence interval [CI]) and 33.2 ± 17.3% of wt, respectively (Fig. 3E). Quantitative PCR indicated that the amount of Bcl11b transcripts was comparable with that observed in wt and the three PD mutant strains (Fig. 3F), suggesting that the reduced amounts of Bcl11bE4PM and Bcl11bE2*4PM proteins were caused by posttranslational mechanisms. Therefore, we evaluated the t1/2s of wt and mutant Bcl11b proteins using CHX, an inhibitor of translational elongation (19). Freshly isolated thymocytes were treated with CHX for 4 h, and the amounts of Bcl11b proteins were measured by immunoblotting. The estimated t1/2 of wt protein was 13.7 ± 1.1 h (mean ± 95% CI), and that of Bcl11bE2PM was 13.7 ± 1.2 h (Fig. 4A). In contrast, Bcl11bE4PM and Bcl11bE2*4PM mutant proteins showed shorter mean lifetimes, and their t1/2s were 5.4 ± 0.6 and 4.5 ± 0.9 h, respectively (Fig. 4A). This result indicated that Bcl11bE4PM and Bcl11bE2*4PM proteins were degraded more rapidly than wt Bcl11b protein. Because the ubiquitin-proteasome pathway is known to be involved in protein degradation (20), we next investigated whether this pathway is involved in the degradation of Bcl11b proteins by a proteasome inhibitor, MG-132 (21). Thymocytes from Bcl11b+/+ and Bcl11bE2*4PM/E2*4PM mice were treated with MG-132 for 6 h, and Bcl11b levels were analyzed by immunoblotting. Treatment with MG-132 considerably increased the Bcl11b protein levels in both Bcl11b+/+ and Bcl11bE2*4PM/E2*4PM thymocytes (Fig. 4B), revealing the involvement of the proteasome pathway in the degradation of Bcl11b protein. These findings suggest that phosphorylation of Bcl11b ensures protein stability by counteracting proteasome-mediated protein degradation. The increase in Bcl11b protein levels by MG-132 treatment in Bcl11b+/+ cells suggests that a proportion of Bcl11b protein in a cell is likely to be phosphorylated at steady state.

FIGURE 4.

Acceleration of proteasome-dependent degradation of Bcl11b protein by Bcl11bE4PM and Bcl11bE2*4PM mutations. (A) CHX chase assay was performed with thymocytes from indicated genotypes to calculate the t1/2 of wt and PD mutant Bcl11b proteins. Three independent experiments were performed. Cells were treated with CHX for 4 h, and the amount of Bcl11b protein was determined by immunoblotting. Error bars indicate 95% CI. (B) Proteasome-mediated degradation of Bcl11b protein. Thymocytes from Bcl11b+/+ or Bcl11bE2*4PM/E2*4PM mice were treated with DMSO or MG-132. After 6 h, cells were harvested, and the amount of Bcl11b protein was measured by immunoblotting. Three independent experiments were performed. Error bars indicate 95% CI. *p < 0.05, **p < 0.005, ***p < 0.0005.

FIGURE 4.

Acceleration of proteasome-dependent degradation of Bcl11b protein by Bcl11bE4PM and Bcl11bE2*4PM mutations. (A) CHX chase assay was performed with thymocytes from indicated genotypes to calculate the t1/2 of wt and PD mutant Bcl11b proteins. Three independent experiments were performed. Cells were treated with CHX for 4 h, and the amount of Bcl11b protein was determined by immunoblotting. Error bars indicate 95% CI. (B) Proteasome-mediated degradation of Bcl11b protein. Thymocytes from Bcl11b+/+ or Bcl11bE2*4PM/E2*4PM mice were treated with DMSO or MG-132. After 6 h, cells were harvested, and the amount of Bcl11b protein was measured by immunoblotting. Three independent experiments were performed. Error bars indicate 95% CI. *p < 0.05, **p < 0.005, ***p < 0.0005.

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Bcl11b is essential for T cell lineage commitment (6–8) and TCR-mediated cell fate determination processes in the thymus, such as positive selection of thymocytes and appropriate expression of lineage-specifying transcription factors such as Zbtb7b (also known as ThPOK), Runx3, and Foxp3 (14, 22, 23). To investigate whether blockage of phosphorylation of Bcl11b protein has any impact on its function in regulating thymocyte differentiation, we performed flow-cytometric analysis. There were no remarkable changes in thymocyte cellularity in any of the Bcl11b-PM mice (Fig. 5A). Analysis of thymocyte subsets defined by CD4 and CD8α coreceptor expression revealed a normal proportion of DN, double-positive (DP), and single-positive (SP) subsets (Fig. 5B, 5C). Expression of CD44 and CD25 also showed that early T cell development at the DN1–DN4 stages was unaffected in the three mutant strains (Fig. 5B, 5C). The frequency and absolute numbers of mature thymocytes, defined as CD24TCRβ+ cells in these strains, were also comparable with those in wt control mice (Fig. 5B, 5C). It has been reported that Bcl11b is necessary for regulatory T cell development in the thymus, and that CD25 is a marker of regulatory T cells among CD4 SP thymocytes (22). Our analysis of CD25 expression on CD4 SP thymocytes revealed no difference in the percentage of CD25+ cells between wt and mutant strains (Fig. 5B, 5C). It has been shown that hypomorphic Bcl11bS826G mutation, accompanied by Bcl11bΔ mutation, induces the development of innate memory-like CD8 SP thymocytes that are characterized by high CD122 and CD44 expression (13). In mature CD8 SP thymocyte populations of Bcl11bE4PM/E4PM and Bcl11bE2*4PM/E2*4PM mice, the frequency of the CD122hi CD44hi subset increased marginally. Thus, unlike the Bcl11bS826G mutation, the Bcl11bE2*4PM mutation prevented the generation of innate memory-like CD8 SP thymocytes (Fig. 5B, 5C). These observations indicate that dephosphorylation of multiple S/T residues of Bcl11b protein by a genetic manipulation has little or no impact on primary T cell development in the thymus.

FIGURE 5.

Normal development of T cells in the thymus. Flow-cytometric analyses of thymocytes were performed (Bcl11b+/+ [n = 10], Bcl11bE2PM/E2PM [n = 6], Bcl11bE4PM/E4PM [n = 3], and Bcl11bE2*4PM/E2*4PM [n = 5]). (A) Absolute numbers of total thymocytes from each genotype are shown. Error bars indicate 95% CI. (B) Percentages of indicated thymocyte subsets in indicated thymocyte population are shown. Error bars indicate 95% CI. (C) Representative plots of flow-cytometric analyses are shown. Numbers in the plots indicate percentages of each region.

FIGURE 5.

Normal development of T cells in the thymus. Flow-cytometric analyses of thymocytes were performed (Bcl11b+/+ [n = 10], Bcl11bE2PM/E2PM [n = 6], Bcl11bE4PM/E4PM [n = 3], and Bcl11bE2*4PM/E2*4PM [n = 5]). (A) Absolute numbers of total thymocytes from each genotype are shown. Error bars indicate 95% CI. (B) Percentages of indicated thymocyte subsets in indicated thymocyte population are shown. Error bars indicate 95% CI. (C) Representative plots of flow-cytometric analyses are shown. Numbers in the plots indicate percentages of each region.

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In the spleen of Bcl11bE2PM/E2PM, Bcl11bE4PM/E4PM, and Bcl11bE2*4PM/E2*4PM mice, frequencies and numbers of CD3ε+TCRβ+ T cells were comparable with those in the spleen of wt control mice (Fig. 6A, 6B). The proportions of CD4+ Th and CD8+ killer T cell subsets were also comparable between wt control and Bcl11bE2PM/E2PM mice (Fig. 6A, 6C). Furthermore, the ratio of naive versus memory-activated T cell subsets, assessed by CD62L and CD44 expression, was similar in both CD4+ and CD8+ T cell populations of wt and PD mutant strains (Fig. 6A, 6D). These results indicate that phosphorylation of Bcl11b is not necessary for its function in the homeostasis of conventional T cells.

FIGURE 6.

Comparable proportion of peripheral T cells and differentiation into effector Th cell subsets between wt and mutant mice. (AD) Flow-cytometric analyses of splenic T cells were performed (Bcl11b+/+ [n = 8], Bcl11bE2PM/E2PM [n = 5], Bcl11bE4PM/E4PM [n = 3], and Bcl11bE2*4PM/E2*4PM [n = 4]). (A) Representative plots of flow-cytometric analyses of splenic Th and killer T cells are shown. Numbers in the plots indicate percentages of each region. Total splenic T cells were defined as CD3ε+TCRβ+ cells. (B) Absolute numbers and percentages of total splenic T cells from each genotype are shown. Error bars indicate 95% CI. (C) Percentages of CD4+ Th and CD8α+ killer T cells in the spleen are shown. Error bars indicate 95% CI. (D) Percentages of CD62L/CD44 expressing Th and killer T cell subsets are shown. Error bars indicate 95% CI. (E and F) In vitro differentiation of Bcl11b+/+:OT-II and Bcl11bE2*4PM/E2*4PM:OT-II splenic CD4 T cells into Th1, Th2, Th17, and iTreg subsets. Representative plots of intracellular staining out of three experiments are shown in (E), and percentages of cytokine-producing cells and Foxp3-expressing cells are shown as average value ± 95% CI of three independent experiments in (F).

FIGURE 6.

Comparable proportion of peripheral T cells and differentiation into effector Th cell subsets between wt and mutant mice. (AD) Flow-cytometric analyses of splenic T cells were performed (Bcl11b+/+ [n = 8], Bcl11bE2PM/E2PM [n = 5], Bcl11bE4PM/E4PM [n = 3], and Bcl11bE2*4PM/E2*4PM [n = 4]). (A) Representative plots of flow-cytometric analyses of splenic Th and killer T cells are shown. Numbers in the plots indicate percentages of each region. Total splenic T cells were defined as CD3ε+TCRβ+ cells. (B) Absolute numbers and percentages of total splenic T cells from each genotype are shown. Error bars indicate 95% CI. (C) Percentages of CD4+ Th and CD8α+ killer T cells in the spleen are shown. Error bars indicate 95% CI. (D) Percentages of CD62L/CD44 expressing Th and killer T cell subsets are shown. Error bars indicate 95% CI. (E and F) In vitro differentiation of Bcl11b+/+:OT-II and Bcl11bE2*4PM/E2*4PM:OT-II splenic CD4 T cells into Th1, Th2, Th17, and iTreg subsets. Representative plots of intracellular staining out of three experiments are shown in (E), and percentages of cytokine-producing cells and Foxp3-expressing cells are shown as average value ± 95% CI of three independent experiments in (F).

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We next investigated whether other T cell subsets, γδT cells, iNKT cells, and IELs, were affected by the PM mutations. Analysis of γδT cells in the thymus, spleen, liver, or intestine showed that their proportions were comparable between Bcl11b+/+ and Bcl11bE2*4PM/E2*4PM mice (Supplemental Fig. 4A–C). In addition, detection of liver iNKT cells with αGalCer-loaded CD1d-tetramer demonstrated that iNKT populations were intact in Bcl11bE2*4PM/E2*4PM mice (Supplemental Fig. 4C). Analyses of IELs revealed normal frequency of total αβT cells, and we found comparable proportions of CD8α+ cytotoxic CD4 and CD8αα+ T cells between Bcl11b+/+ and Bcl11bE2*4PM/E2*4PM mice (Supplemental Fig. 4D). These observations indicate that PM mutation has minimal effects on the development of nonconventional T cell subsets.

Phosphorylation of S/T residues of Bcl11b protein was shown to occur upon TCR stimulation (10), indicating toward the possibility that Bcl11b phosphorylation could be involved in regulating effector CD4 T cell differentiation when T cells encounter Ags at the periphery. Therefore, we examined the in vitro differentiation of CD4+ naive T cells obtained from Bcl11bE2*4PM/E2*4PM mice into effector Th cells. To this end, Bcl11bE2*4PM/E2*4PM mice were crossed with OT-II transgenic mice that expressed the TCR recognizing chicken OVA 323–339 peptide in the context of MHC class II molecules (24). OT-II CD4 naive T cells were stimulated by OVA 323–339 peptide under specific cytokine combinations to drive Th1, Th2, Th17, and Treg cell differentiation (25, 26). Under Th1 conditions, the extent of differentiation into IFN-γ–producing Th1 cells was comparable between OT-II wt and Bcl11bE2*4PM/E2*4PM cells. Differentiation into IL-4–producing Th2 and IL-17–producing Th17 cells was also not affected by the Bcl11bE2*4PM mutation, and differentiation into Foxp3-expressing Treg cells was equally induced between OT-II wt and Bcl11bE2*4PM/E2*4PM cells (Fig. 6E, 6F). Overall, phosphorylation of 23 S/T residues of Bcl11b is dispensable for in vitro differentiation into effector Th cell subsets.

Because Bcl11b was reported to be critical for the airway Th2 responses (27), we next investigated the requirement of Bcl11b phosphorylation in Th2 immune responses in vivo. To this aim, we challenged the Bcl11bE2*4PM/E2*4PM mice against the A. alternata–induced lung inflammation model. For this study, we injected an extract of A. alternata intranasally into Bcl11b+/+ and Bcl11bE2*4PM/E2*4PM mice every 3 d, and 2after 24 h after final challenge (day 7), we measured cytokine expression in lung-derived Th2 cells and group 2 innate lymphoid cells (ILC2s) and BALF eosinophil infiltration. At day 7, we found that the absolute numbers of lung-derived Th2 cells (defined as CD45+Thy1.2+CD4+TCRβ+Gata3+ST2+ cells) and ILC2s (defined as CD45+Thy1.2+CD4TCRβGata3+ST2+ cells) were comparable between Bcl11b+/+ and Bcl11bE2*4PM/E2*4PM mice (Fig. 7A, 7B). Moreover, the intracellular staining of IL-4 and IL-5 further revealed these lung-derived Th2 cells from both wt and mutant mice equivalently produced these cytokines (Fig. 7A, 7C). Infiltration of eosinophils, which is mediated mainly by IL-5 from ILC2s, is a hallmark for airway inflammation by A. alternata (Fig. 7A, 7C). We were able to detect the emergence of BALF-derived Th2 cells and eosinophils at 7 d after A. alternata extract injection, which were again comparable between Bcl11b+/+ and Bcl11bE2*4PM/E2*4PM mice (Fig. 7D, 7E). In line with these observations, IL-5 production from ILC2s of Bcl11bE2*4PM/E2*4PM mice was similar to that from control cells (Fig. 7A, 7C). These observations indicated that the airway inflammation mediated by Th2 response was not impaired by the PM mutation.

FIGURE 7.

Major phosphorylation on Bcl11b is dispensable for in vivo type 2 immune responses. The respiratory Th2 responses in Bcl11b+/+ and Bcl11bE2*4PM/E2*4PM mice were investigated by the administration of A. alternata extract. (A) Representative plots of flow-cytometric analyses of lung Th2 cells and ILC2s in CD45+Thy1.2+ population are shown. Numbers in the plots indicate percentages of each region. Th2 cells and ILC2s were defined as CD4+TCRβ+Gata3+ST2+ cells and CD4TCRβGata3+ST2+ cells, respectively, and the production of IL-4 and IL-5 was investigated by intracellular staining. (B) Absolute numbers of lung Th2 cells and ILC2s are shown as average value ± SD of three independent experiments. (C) Percentages of IL-4+IL-5 and IL-4+IL-5+ Th2 cells, and IL-4+IL-5+ and IL-4IL-5+ ILC2s are shown as average value ± SD of three independent experiments. (D) Representative plots of flow-cytometric analyses of BALF Th2 cells and eosinophils are shown. Numbers in the plots indicate percentages of each region. Eosinophils were defined as CD45+CD11cSiglec-F+ cells. (E) Absolute numbers of BALF Th2 cells and eosinophils are shown as average value ± SD of three independent experiments.

FIGURE 7.

Major phosphorylation on Bcl11b is dispensable for in vivo type 2 immune responses. The respiratory Th2 responses in Bcl11b+/+ and Bcl11bE2*4PM/E2*4PM mice were investigated by the administration of A. alternata extract. (A) Representative plots of flow-cytometric analyses of lung Th2 cells and ILC2s in CD45+Thy1.2+ population are shown. Numbers in the plots indicate percentages of each region. Th2 cells and ILC2s were defined as CD4+TCRβ+Gata3+ST2+ cells and CD4TCRβGata3+ST2+ cells, respectively, and the production of IL-4 and IL-5 was investigated by intracellular staining. (B) Absolute numbers of lung Th2 cells and ILC2s are shown as average value ± SD of three independent experiments. (C) Percentages of IL-4+IL-5 and IL-4+IL-5+ Th2 cells, and IL-4+IL-5+ and IL-4IL-5+ ILC2s are shown as average value ± SD of three independent experiments. (D) Representative plots of flow-cytometric analyses of BALF Th2 cells and eosinophils are shown. Numbers in the plots indicate percentages of each region. Eosinophils were defined as CD45+CD11cSiglec-F+ cells. (E) Absolute numbers of BALF Th2 cells and eosinophils are shown as average value ± SD of three independent experiments.

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Bcl11b has been shown to be crucial for T lymphocytes lineage commitment and positive selection of thymocytes upon receiving TCR signals. PTMs such as phosphorylation and SUMOylation of proteins are known to regulate different aspects of proteins, such as activation, molecular interactions, and stabilization. It has been reported that Bcl11b undergoes phosphorylation at 25 S/T residues upon PMA/ionomycin stimulation and SUMOylation at two K residues (K679 and K877) in thymocytes (10). In this study, we showed that dephosphorylation of Bcl11b with AP shifted the electrophoretic mobility of wt Bcl11b protein, and LC-MS/MS analysis confirmed that Bcl11b is partly phosphorylated in thymocytes at steady state (Fig. 3A, 3D, Supplemental Fig. 3). The phosphorylation of Bcl11b has been reported to be mediated by MAPK upon TCR stimulation (28). Most of the phosphorylation sites on Bcl11b are found in -x-S/T-P-x- motifs (NP_001073352), which are targeted by cyclin-dependent kinase, MAPK, glycogen synthase kinase and CDC-like kinase (29). In addition, the Ras/MAPK pathway is involved in the positive selection of thymocytes (30). Based on these observations, we hypothesized that the phosphorylation state of Bcl11b plays an important role in transmission of TCR signaling into mechanisms that control cell fate. To test this hypothesis, we mutated the majority of the phosphorylation sites of Bcl11b using genetic approaches. Among 25 phosphorylation sites that were reported by Zhang et al. (10), our LC-MS/MS analysis detected the phosphorylation on 21 residues and seven new putative phosphorylation acceptors, all of which were S/T residues (Fig. 3D, Supplemental Fig. 2B). LC-MS/MS analysis also confirmed that phosphorylation on those major S/T sites was lost in Bcl11bE2*4PM protein (Fig. 3D, Supplemental Fig. 2B). Interestingly, in addition to the unmanipulated S128 and S169 residues, we observed the noncanonical phosphorylation on S487 and S725 residues specifically in Bcl11bE2*4PM protein with >99% probability (Fig. 3D, Supplemental Fig. 2B). Thus, the major physiological S/T phosphorylation was lost in Bcl11bE2*4PM protein. Surprisingly, the efficiency of positive selection and the Th/cytotoxic T cell lineage choice were not impaired by extensive mutagenesis of multiple S/T residues of Bcl11b protein. Our findings revealed that phosphorylation of 23 S/T residues in Bcl11b, which is mediated by the Ras/MAPK pathway, at least in culture, is dispensable for the function of Bcl11b in primary T cell development in the thymus. Importantly, the Bcl11bE2*4PM protein still supported the differentiation of effector Th cells in vitro (Fig. 6) and the airway Th2 response against A. alternata (Fig. 7). These observations suggested that phosphorylation on the major S/T sites might be dispensable for Bcl11b function in regulating Th2 response. Although it is not clear whether Bcl11b is phosphorylated in ILC2s, our results obtained by the A. alternata model suggest that phosphorylation on major S/T residues, if any, are dispensable for Bcl11b function in ILC2s. However, current reports have also shown the importance of Bcl11b in cytotoxic T cell expansion and function of cytotoxic T cells (31). Hence further analyses are necessary to fully understand the role of phosphorylation of Bcl11b in effector T cell functions.

Immunoblotting experiments detected multiple faint signals above the two major signals of Bcl11b forms (Fig. 3A). Based on previous studies and the immunoblotting result (10, 17), these major signals were concluded to correspond to un-SUMOylated Bcl11b proteins, whereas the faint signals represent SUMOylated proteins (Fig. 3A, 3B). Our analysis showed that the proportion of these SUMOylated Bcl11b was higher in Bcl11bE4PM/E4PM and Bcl11bE2*4PM/E2*4PM thymocytes. Approximately 40% of the total Bcl11b was SUMOylated in these genotypes, whereas in the case of wt and Bcl11bE2PM/E2PM mice, only 15% of the protein was SUMOylated (Fig. 3B). Thus, decreased phosphorylation caused by A replacement might facilitate SUMOylation of Bcl11b, which is consistent with a previous study showing reciprocal regulation between phosphorylation and SUMOylation of Bcl11b in DN thymocytes (17).

Computational studies using the Protein DisOrder prediction System (http://prdos.hgc.jp/cgi-bin/top.cgi) (32) predicted that phosphorylation acceptor S/T residues are predominantly located in disordered regions (Supplemental Fig. 2A), suggesting that these residues might control molecular interaction. It has been suggested that the balance between phosphorylation and SUMOylation is critical for controlling the interactions of Bcl11b with NuRD, a chromatin remodeling complex (10, 33, 34). Further, Dubuissez et al. (35) reported that S2 within the N-terminal MSRRKQ motif in Bcl11b can be phosphorylated by protein kinase C, and that this phosphorylation negatively controls the recruitment of the NuRD complex without affecting SUMOylation. Although the NuRD complex was shown to regulate the expression of T cell–related genes such as Cd4 and Il2r (36, 37), analysis of the Bcl11b-PM mutant strains did not reveal impaired expression of these surface molecules (Fig. 5C). This indicates toward a possibility that loss of phosphorylation at S/T residues encoded by exons 2 and 4 might not affect the function of NuRD complexes. It would be interesting to examine whether Bcl11b phosphorylation at S2 or other S/T residues will influence the interaction with the NuRD complex.

We showed that the Bcl11bE4PM and Bcl11bE2*4PM mutant proteins were unstable because their calculated t1/2s were shorter than those of the wt Bcl11b protein (Fig. 4A). Previously, the t1/2 of Bcl11b evaluated in the T cell line was reported to be ∼4 h (38). However, in this study, the t1/2 evaluated in primary cells was ∼14 h. This discrepancy might stem from the rapid proliferation of the T cell line established in vitro. Computational predictions on the basis of amino acid sequences indicated that instabilities of the wt and Bcl11bE2*4PM proteins were comparable, instability index = 62.8 and 60.0, respectively (https://web.expasy.org/protparam/) (39). No additional typical proteolytic sites were predicted in the mutant Bcl11bE2*4PM protein. Previous reports have reported the ubiquitination and degradation of Bcl11b under prolonged PMA/ionomycin stimulation (10). Given the recovery of protein stability by MG-132 treatment, the mutant Bcl11bE2*4PM protein is degraded by the proteasome pathway. These results suggest that the shorter t1/2 obtained after A replacement likely stems from the loss of phosphorylation and not from acquiring additional proteolytic sites. Other cases in which phosphorylation upregulates protein ubiquitination and downregulates the subsequent protein degradation have also been reported (40). Phosphorylation of S/T residues was shown to generate a phosphodegron that promotes ubiquitination of nearby K residues by recruiting ubiquitin ligase. Further, phosphorylation of S residue on the d-box motif was proposed to increase the stability of proteins by inhibiting the interaction of anaphase promoting complex/cyclosome ubiquitin ligase (41). In the case of Bcl11b protein, it is possible that phosphorylation of S/T residues could suppress the proteasome-dependent degradation, although our results do not exclude the possibility that conformational changes caused by extensive A replacement destabilize the mutant Bcl11bE2*4PM protein. Both Bcl11bE4PM/E4PM and Bcl11bE2*4PM/E2*4PM mice displayed growth retardation from 2 to 4 wk of age (Fig. 2B, 2C). Nevertheless, these mutant mice appear to be healthy, can survive for >1 y, and their body weight can normalize at their adult stages (Fig. 2B). Heterozygous knockout of Bcl11b in mice also led to weight loss, which was partly caused by insufficient nutritional intake (9, 16). Therefore, a possible reason for the growth retardation observed in Bcl11bE4PM/E4PM and Bcl11bE2*4PM/E2*4PM mice could be the lower levels of Bcl11b protein.

Although extensive replacement of S/T residues with A in the Bcl11b protein did not considerably impair the functions of Bcl11b protein, two missense variants in the human BCL11B gene have been reported to cause immunological and/or neurologic defects (42–44). A de novo missense mutation of the human BCL11B gene, p.N441K, was identified in a patient with SCID (43). In this patient, the levels of both CD4 and CD8 T cells in the peripheral circulation were substantially low, and functional assays suggested a dominant-negative function of the mutant protein (43). Another missense mutation, BCL11b p.N807K, also impaired development of T cells and ILC2s. Bcl11b is also required for the development of ILC2s in mice (44, 45). The N441 and N807 residues are located in the second (aa 427–454) and fourth (aa 796–823) C2H2 ZnF domains of BCL11B (NP_612808), respectively. In a N-ethyl-N-nitrosourea mutagenesis study in mice, Hirose et al. (13) reported that the Bcl11bS826G allele impaired positive selection and caused the emergence of innate memory-like CD8 SP thymocytes. The S826 residue is located in the fifth C2H2 ZnF (aa 814–843) of murine Bcl11b (NP_001073352) and is not likely to be a phosphorylation site (Supplemental Fig. 2B) (10). These observations indicate that missense mutations that alter the conformation of the ZnF domains impair the function of BCL11B/Bcl11b. Thus, the ZnF domains are crucial for Bcl11b function and not the phosphorylation of S/T residues.

Although phosphorylation of the S/T residues is dispensable for Bcl11b function, it is possible that its function is regulated by other PTMs. After identifying the PTMs of Bcl11b in cell lines, analysis of mouse lines expressing mutant Bcl11b lacking these PTMs is the most reliable approach to unraveling the relevance of PTMs. Analysis of mouse lines that harbor mutations corresponding to pathological missense variants of human transcription factors serves as a powerful tool for understanding the pathogenesis of human diseases, as was recently exemplified by a report on the IKZF3 p.G159R mutation, which was detected in the family of a patient with B cell deficiency (46).

The authors have no financial conflicts of interest.

We thank Dr. Y. Katsuragi (Niigata University) for providing Bcl11b targeting vector. We thank Y. Taniguchi and C. Miyamoto (RIKEN Yokohama) for technical support and genotyping of mice. We appreciate members from the Animal Facility Group at RIKEN center for integrative medical sciences. We also thank our laboratory members for valuable discussions.

This work was supported by the Japan Society for the Promotion of Science KAKENHI 20K07459 (to K.O.), Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research on Innovative Areas 19H04820 (to I.T.), and Joint Usage and Joint Research Programs of the Institute of Advanced Medical Sciences, Tokushima University (to I.T.).

The online version of this article contains supplemental material.

ACN

acetonitrile

AP

alkaline phosphatase

BALF

bronchial alveolar lavage fluid

CHX

cycloheximide

CI

confidence interval

DN

double-negative

ES

embryonic stem

αGalCer

Α-galactosyl ceramide

gRNA

guide RNA

IEL

intraepithelial lymphocyte

ILC2

group 2 innate lymphoid cell

iNKT

invariant NKT

LC

liquid chromatography

neor

neomycin-resistant gene

PM

phosphorylation site mutation

PTM

posttranslational modification

SP

single-positive

Treg

regulatory T

wt

wild-type

ZnF

zinc-finger

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Supplementary data