Identification of a novel enhancer essential for Satb1 expression in T_H2 cells and activated ILC2s

Mice

The Satb1^Venus allele was generated by knock-in insertion of the Venus open reading frame downstream of transcription start site (exon2) of the Satb1 gene. For this aim, a BAC clone B6Ng01-312L13, which included the 5′ area of the Satb1 gene, was purchased from RIKEN BRC. A 13-kb genomic region harboring the ATG start codon was subcloned into the pBlueScript II vector (Stratagene). The DNA fragment harboring partial exon2 sequences and Venus cDNA sequences were generated by overlapping the PCR technique and was used to construct a targeting vector. The targeting vector was transfected into murine M1 ES cells as previously described (28). G418-resistant ES clones were screened for homologous recombination event between the target vector and the Satb1 gene. Appropriate ES clones were then used to generate chimera mice by ES aggregation. F1 founders from the chimera mice carrying the Satb1^Venus allele were selected for establishing mouse line and were analysed. Satb1^Flx CD4-cre mice were previously described in (7). Satb1^+/Venus-Δa (or Satb1^Venus-ΔEth2) and Satb1^+/Venus-Δb mice were generated by co-injection of Cas9-mRNA with sgRNAs into Satb1^+/Venus fertilised eggs that were generated by in vitro fertilisation with sperm from a Satb1^Venus/Venus male mouse and Satb1^+/+ oocytes. The F0 founders that had deleted Satb1-a or Satb1-b regions were then crossed with C57/B6N mice and F1 founders that deleted Satb1-a or Satb1-b on the Satb1^Venus allele were selected as heterozygous mice with the Satb1^+/Venus-Δa or Satb1^+/Venus-Δb genotype. Among the two and three F1 Satb1^+/Venus-Δa and Satb1^+/Venus-Δb founders, we chose one line as a representative for Satb1^Venus-Δa and Satb1 ^Venus-Δb and were examined with littermate controls. Similarly, C57/B6N Satb1^Δa (Satb1^ΔEth2) mice were generated by co-injection of Cas9-mRNA with Satb1-a gRNA into C57Bl/6 fertilised eggs. Sequences for sgRNA are as follows: Satb1^Δa sgRNAs 5′-TCAACATCAGAATTTCT-3′ and 5′-CAGTCAACATCAGAATTTCT-3′; and Satb1^Δb sgRNAs 5′-ACACACACTGTCTGTTGTGC-3′ and 5′-GCTGCCTGCTTTTACATATC-3′. All mice were maintained at the RIKEN Center for Integrative Medical Sciences. The animal protocol was approved by the Institutional Animal Care and Use Committee of RIKEN Yokohama Branch (2020-026). For all experimental procedures, at least two to three mice were used per experiment and are shown either as a representative or as a summary of these biological experiments.

Immunohistochemistry

50,000 of thymocytes from Satb1^+/Venus mice were resuspended in 0.1 ml RPMI-1640 medium (10% FBS) and were mounted on a poly-L-lysine-coated glass slide (REF: 26414; Polysciences, Inc.) and incubated at 37°C for 2 h. The attached cells were gently washed three times in PBS. The cells were fixed with 4% PFA at room temperature for 10 min and then washed three times in PBS. The cells were permeabilized with 70% ethanol and kept at −20°C until analysis. On the day of the analysis, thymocytes were stained with 1/100 of DAPI (ab228549; Abcam) at room temperature, for 30 min, and washed three times in PBS. Cover slips were mounted on top of the thymocytes with Fluoromount-G mounting medium (00-4958-02; Thermo Fisher Scientific) and sealed with Biotium CoverGrip Coverslip Sealant (23005; REF). Confocal microscopic images were obtained with TCS-SP5 (Leica Microsystems) using 40x objective.

Ex vivo cell preparation for flow cytometry

Thymus, spleen, and peripheral lymph nodes (axillary, inguinal, and cervical) were removed from mice at 4–8 wk of age and were mashed through a 70-μm cell strainer in a Petri dish to make single-cell suspensions. For the elimination of red blood cells, splenocytes were treated with ACK lysis buffer (Gibco A1049201, 2.5 ml per spleen) for 3 min and pelleted by centrifugation at 300g at 4°C for 5 min. Supernatants were discarded and cell pellets were resuspended into RPMI-1640 (2% FBS). All cell suspensions were maintained at 4°C for flow cytometry analyses.

For the preparation of IEL, the small intestine was cut into three sections and Peyer Patches were removed. Each section was cut longitudinally and washed twice with HBSS (5% of FCS). Sections were further cut into 0.5-cm pieces and put into a 50 ml conical tube containing 25 ml HBSS (5% of FCS). The tissues were then placed in a bioshaker (TAITEC, BR-43FL) at 37°C, at 250 rpm for 15 min. The supernatant was transferred into a new 50 ml tube, through a metal mesh, and cells were pelleted down by centrifugation at 300g at 4°C for 5 min. The supernatant was discarded. The cell pellet was then resuspended in 8.5 ml of HBSS + 40% Percoll and layered onto 2 ml HBSS+ 70% Percoll in a 15 ml falcon tube. The cells were then centrifuged at 860g for 25 min at room temperature with brakes off. IEL were collected in the interphase between 40% and 70% Percoll and washed twice with HBSS (5% of FCS). IEL were then subjected to flow cytometric analyses.

For the preparation of lung cell suspensions, the lungs were first perfused with 20 ml of PBS through the left ventricle of the heart. Perfused lungs were dissected out and minced up before incubation in RPMI-1640 (2% FBS) + 0.3 mg/ml collagenase IV (C-5138; Sigma-Aldrich) + 0.3 mg/ml of DNase I (043-26773; Wako) at 37°C for 45 min with continuous shaking. To homogenize the samples, the digested lung tissues were mashed in a 70-μm cell strainer in a Petri dish and cell suspensions were then pelleted by centrifugation at 300g at 4°C for 5 min. For the elimination of debris, the cell pellets were resuspended in 10 ml RPMI (2% FBS) + 30% Percoll and separated by centrifugation at 860g at room temperature for 20 min with brakes off. Debris in the upper phase were aspirated off and cell pellets were resuspended in 5 ml RPMI-1640 (2% FBS). The cells were washed and pelleted by centrifugation at 300g at 4°C for 5 min and were then subjected for analysis.

Flow cytometry

Cells were stained with the following fluorophore-conjugated antibodies: B220 (RA3-6B2) CD4 (RM4-5), CD8a (53-6.7), CD11B (M1/70), CD11C (HL3), CD19 (1D3), CD24 (M1/69), CD25 (PC61.5), CD44 (IM7), CD45 (30-F11), CD62L (MEL-14), CD69 (H1.2F3), FOXP3 (FJK-16s), GATA3 (L50-823), Gr1 (RB6-8C5), IL-4 (11B11), IL-5 (TRFK5), IFN-γ (XMG1.2), Ly6C (AL-21), Ly6G (1A8), NK1.1 (PK136) Siglec-F (E13-161.7), ST2 (U29-93) TCRβ (H57-597), TCRγδ (GL3), and Ter119 (TER-119). Murine CD1d dimmer XI (557599; BD-Bioscience) loaded with α-GalCer was used to define iNKT cells. For flow cytometric analyses 1–2 × 10⁶ cells were washed out of complete medium, by centrifugation at 300g at 4°C for 5 min and resuspended in FACS buffer (PBS + 1% FBS and 0.05% NaN₃). For the analyses of extracellular marker on live cells, cell suspensions from spleen, pLNs, IEL, and lungs were treated with rat α-mouse CD16/CD32 (BD Biosciences) for 10 min at 4°C. Fluorophore antibodies for extracellular markers were then added and stained for 30 min at 4°C. After incubation, cells were then pelleted by centrifugation at 300g at 4°C for 5 min and resuspended in FACS buffer containing 7AAD/DAPI cell viability dye. For analyses of both extracellular and intracellular markers, cell suspensions of all tissues were stained with fixable live/dead cells (65-0866-18; Ebiosciences) for 10 min before rat α-mouse CD16/CD32 treatment and extracellular marker staining. Using the FOXP3 staining buffer kit (00-5523-00; Ebiosciences), stained cells were then fixed, permeabilised, and stained for intracellular transcription factors as per the manufacturer’s instructions. All cytometry analyses were performed using a FACS CANTO II (BD-Bioscience) and data were analysed using FlowJo (Tree Star) software.

In vitro CD4 T helper differentiation

Naïve CD4 T cells were pooled from spleen and peripheral lymph nodes. The spleen and peripheral lymph nodes (axillary, inguinal, and cervical) were removed from mice at 6–10 wk of age and were mashed through a 70-μm cell strainer in a Petri dish to make single-cell suspensions. Splenocytes were the treated with ACK lysis buffer (Gibco A1049201, 2.5 ml per spleen) for 3 min before pelleting (by centrifugation at 300g at 4°C for 5 min) and resuspension into RPMI-1640 (2% FBS). Naïve CD4 T cells from either sorted by FACS Aria (sorted for CD4⁺CD25⁻CD62L^hiCD44⁺) or isolated by using EasySep mouse naïve CD4 T cell isolation kit (ST-19765). Naïve T cells were resuspended in complete medium (KOHJIN BIO DMEM-H [16003016] + 10% FBS) and activated in the presence of 2 μg/ml of anti-CD3 (REF: 553058; BD Pharmingen) and 2 μg/ml of anti-CD28 (REF: 553295; BD Pharmingen) (precoated in a rounded 96-well plate) in T_H0 (5 μg/ml of anti-IFNγ [REF: 16-741185; Invitrogen], 5 μg/ml of anti-IL-12/23 [REF: 505308; BioLegend], 5 μg/ml of anti-IL-4 [REF: 554385; BD Pharmingen] and 10 ng/ml of IL-2 [404-ML-010; R&D systems]), T_H1 (5 μg/ml of anti-IL-4, 10 ng/ml of IL-2 and 10 ng/ml of IL-12 [419-ML-010; R&D systems]), T_H2 (5 μg/ml of anti-IFN- γ, 5 μg/ml of anti-IL-12/23,10 ng/ml of IL-2, and 10 ng/ml of IL-4 [402-ML-020; R&D systems]), T_H17 (R and D systems, CDK017, as per the manual’s instructions), and iTreg (5 μg/ml of anti-IFNγ, 5 μg/ml of anti-IL-12/23, 5 μg/ml of anti-IL-4, 2 ng/ml of IL-2 and 3 ng/ml of TGFβ [7666-MB-055; R&D systems]) conditions. Naïve CD4 cells under T_H0, T_H1, and T_H2 conditions were activated were 48 h and were further maintained in T_H0/1/2 polarising conditions for another 3–5 d. Naïve CD4 cells under T_H17 and iTreg conditions were activated for 72 h and were further maintained in T_H17/Treg conditions for another 3–4 d. To confirm CD4 helper differentiation, 1 × 10⁵ of T_H0, T_H1, T_H2 and were stimulated either with 2 μg/ml of anti-CD3 (plate-bound) or with 100 ng/ml of PMA (P8139; Sigma-Aldrich) and 0.5 μg/ml of ionomycin (I0634-1MG; Sigma-Aldrich), in a rounded 96-well plate for 5 h in the presence of 2 μM of monesin (Cay16488-1; Biomol). T_H17 cells were stimulated as per the manufacturer’s instructions (CDK017; R and D systems). After stimulation, cells were analysed for intracellular expression of IFN-γ, IL-4, and IL-17 by flow cytometry. The in vitro differentiated Tregs were analysed for the intracellular expression of FOXP3 by flow cytometry. After confirmation of CD4 helper differentiation, Satb1 expression levels were analysed by flow cytometry, immunoblotting, and qRT-PCR. Culture supernatants of PMA and ionomycin-treated CD4 T_H0 and CD4 T_H2 cells (in the absence of monesin) were also recovered, flash-frozen in N₂, and stored in –80°C. The quantification of mIL-4 and mIL-5 cytokines in these supernatants was performed by the Laboratory for Immunogenomics (RIKEN, IMS) via Luminex analysis.

Western blotting

T cells in complete medium were pelleted down by centrifugation at 200g for 5 min at 4°C. Supernatants were aspirated, and the cell pellets were resuspended in 1 ml of ice-cold PBS. Cells were transferred into 1.5 ml Eppendorfs and pelleted down by centrifugation at 200g for 5 min at 4°C. The supernatants were aspirated and pellets were washed in 1 ml of ice-cold PBS. The cells were pelleted down by centrifugation at 200g for 5 min at 4°C and supernatants were aspirated. Cells were lysed in lysis buffer (2% SDS in 50 μM Tris–HCL pH.8, supplemented with EDTA-free protease inhibitor, Roche) at cell concentration of 20 × 10⁶/ml and incubated at 95°C for 15 min. Debris were pelleted down by centrifugation at 13,000g for 10 min at room temperature and protein lysates were transferred into new 1.5 ml Eppendorf tubes. Protein lysates were adjusted with 2X laemil sample buffer (Bio-Rad) + 2-β-mercaptoethanol and boiled at 95°C for 10 min. Each lane was loaded with the equivalent of 100,000 T cells and separated by SDS–PAGE in 10% polyacrylamide gels (e-PAGEL, ATTO, EHR-T10L). Proteins were then transferred onto polyvinylidene difluoride membrane (REF:1704156; Bio-Rad) and blots were blocked with TBST (REF: 12749-21, 0.05% vol/vol; Nacalai tesque) + 5% (wt/vol) of milk for 1 h. All antibodies were used accordingly to the manufacturer`s instructions. Blots were then probed with the following primary antibodies in TBST (0.05% vol/vol) + 5% (wt/vol) of milk, overnight at 4°C: SATB1 (ab109122; Abcam), GAPDH (REF: 2275-PC-100; R and D systems) and SMC1 (ab9262; Abcam). The blots were then wash three times in 15–30 ml TBST (0.05% vol/vol) before incubation in HRP-conjugated secondary antibodies (REF:65-6120; Invitrogen) in TBST (0.05% vol/vol) + 5% (wt/vol) of milk. The blots were then wash three times in 15–30 ml TBST (0.05% vol/vol) and chemiluminescence were quantified on the Amersham Imager 680 (GE Healthcare). All immunoblots shown are representative of at least three biological replicates.

qRT–PCR

1 × 10⁶ of T_H0, T_H1 or T_H2 cells were pelleted down by centrifugation at 200g, at 4°C for 5 min. Supernatants were discarded and cell pellets were immediately resuspended in 750 μl of Trizol (REF: 15596026; Ambion). Samples were snap-frozen in liquid N₂. For RNA isolation, cell lysates were thawed, and chloroform added (200 μl per 1 ml of Trizol). RNA samples were extracted as per the manufacturer’s instruction. The samples were vortexed for 20 s and incubated at room temperature for 3 min. The samples were pelleted down by centrifugation at 12,000g at 4°C for15 min. The top aqueous phase containing RNA was transferred into a new tube. RNA samples were purified by using Zymogen RNA clean and concentrator kit (R1017; Zymo research), as per the manufacturer’s instructions. RNA concentrations were obtained on a NanoDrop Spectrophotometer and 1.2 μg of RNA were subjected to cDNA synthesis by using the Super VILO cDNA synthesis kit (REF:11756050; Thermo Fisher Scientific). qRT–PCR was performed using Power up SYBR green master mix (REF: A25742; applied biosystems) on the QuantStudio three Real-Time PCR Systems, accordingly to the manufacturer’s instructions. The following gene expression levels analysed by qRT–PCR are listed in Table 1.

Gm19585 shRNA lentiviral production

The RNAi consortium was used to generate three shRNAs for the knockdown of Gm19585 derived transcripts (see Table 2). shRNA sequences were cloned into the PLKO.gfp cloning vector, which was kindly gifted by Dr. Jun Huh (Harvard University): cloning, transfection, and production of lentiviral particles were performed accordingly to Addgene’s protocol. After screening for shRNA inserts, lentiviruses for individual shRNAs were produced in HEK293T cells plated in six-well plates in antibiotic-free DMEM (Gibco, supplemented with glutamine and 10% FBS). For one transfection assay and using the Fugene HD transfection reagent (REF: E1312; Promega) accordingly to the manufacturer`s instructions, the HEK293T cells were cultured in 10% FBS-containing DMEM and were transfected with 1 μg of shRNA plasmid, 750 ng of psPAX2 packaging plasmid, and 250 ng of pMD2.G envelope plasmids. Packaging and envelope plasmids were obtained from Addgene. Viral supernatants were collected 3 d post transfection and filtered through 0.45 μm low protein-binding filters. Polybrene (Sigma-Aldrich) was added to the viral supernatants at a final concentration of 2 μg/ml, aliquoted, and stored at −80°C.

Gm19585 knock-down in in vitro T_H2 CD4 T cells

CD4 T cells from wild-type mice were isolated from pLNs (axillary, inguinal, and cervical) by using EasySep mouse CD4 T cell isolation kit (ST-19752). Cells were stimulated with 2 μg/ml of immobilised anti-CD3 and anti-CD28 in T_H2 polarising conditions (5 μg/ml of anti-IFN- γ, 5 μg/ml of anti-IL-12/23,10 ng/ml of IL-2, and 10 ng/ml of IL-4) for 24 h. After activation, cells were pelleted down by centrifugation at 200g, at room temperature for 5 min and resuspended in 20 μl of prewarmed PBS. The cells were added to 500 μl of thawed viral supernatants and put into 48-well plates. The cells were centrifuged at 660g at 32°C for 1.5 h and were rested in the tissue culture incubator for 1 h 1 volume of 2X T_H2 cytokines (5 μg/ml of anti-IFN- γ, 5 μg/ml of anti-IL-12/23,10 ng/ml of IL-2, and 10 ng/ml of IL-4) were then added and the cells were incubated for another 48 h pLKO-Gfp (empty) and pLKO-Gfp-Gm19585 shRNA-transduced cells were monitored and T_H2 cytokines were refreshed every second day. CD4⁺GFP⁺ cells were then sorted on 6 d post transduction and immediately lysed in Trizol for qRT–PCR analyses.

ATAC-seq analyses

ATAC-Bigwig files from Immgen database (19) were analysed and visualized on the IGV genome browser. Genomic regions of interest were then further analysed on the USCS genome browser for conservation.

3e HiC data analyses

3e HiC datasets from mESCs (GSM1620000) and CD4 T_H2 cells (GSM1619998) were downloaded from the Gene Expression Omnibus (GEO) site with the accession numbers GSE66343 (22). HiC datasets from thymocytes (GSM5268043) were downloaded from GEO site with the accession number GSE173476 (23). To examine the chromosome structure and topologically associated domain structures surrounding Satb1 and Satb1-a (exon 4 of Gm19585) loci, reads on chromosome 17 were extracted and realigned on mouse genome mm10 using bowtie2 (version 2.4.6). Mapped reads were collected and converted into.hic format using Juicer tools (version 1.22.01, https://github.com/aidenlab/juicer) and visualized using Juicebox (version 1.6, https://github.com/aidenlab/Juicebox/).

H3K27 acetylation and STAT6 ChIP-seq analyses

Publicly available H3K27Ac ChIP-seq datasets from murine CD4⁺FOXP3^- and CD4⁺FOXP3⁺ T cells (8) were used to analyse H3K27Ac peaks at the Satb1-a and Satb1-b loci. The Sequence Read Archive (SRA) files SR5385345 and SRR5385345 were downloaded from SRA website Similarly, STAT6 ChIP-seq in CD4 T_H2 cells (24), was used to analyse STAT6 DNA-binding sites at the Satb1-a locus and the SRA file SRA054075 was downloaded from SRA website. Fastdump was used to convert all SRA files into fasta, which were processed for the removal of adaptor sequences and then realigned to mm10 reference genome using Bowtie2. Samtools was then used to sort and convert the SAM files (output files, generated from the genome alignment) into BAM files. BAM files were then indexed using samtools for the generation of bigwig files. The bigwig files were then visualized on the IGV genome browser.

3C assay

Procedures for 3C are described in our previously published article (29). The primer sequences used for the 3C assay qRT-PCR are provided in Table 3.

A. alternata induced airway inflammation model

8–12 wk old female mice were anesthetized by isofluorane inhalation, followed by intra-nasal or intra-tracheal injection of A.A. extract (ITEA, 10117., 20 μg per head, in 40 μl of PBS) on Day 0, Day 3, Day 6, and/or Day 9. After 24 h post final challenge, naïve and A.A. treated female mice were euthanized by CO₂ inhalation. For cytokine analyses, BAL fluid (BALF) was first collected by intratracheal insertion of a catheter and 1 lavage of 500 μl of HBSS (2% FBS) and transferred into new 1.5 ml Eppendorf tubes. The extracted BALF was immediately pelleted down by centrifugation (300g, 5 min at 4°C) and BALF supernatant were transferred into new 1.5 ml Eppendorf tubes. The pelleted cells were resuspended in 500 μl of HBSS (2% FBS). BALF supernatants were flash-frozen in liquid N₂ and stored in −80°C. Additional two lavages of 500 μl of HBSS (2% FBS) were used to further collect the BALF of naïve and AA challenged mice and were combined with BALF cells from the first lavage, totalling to a final volume of 1.5 ml. BALF cells were kept on 4°C for flow cytometric analyses. Lung cell suspensions from mice were prepared as outlined above. After Percoll gradient centrifugation, CD4 T_H2 cells (CD45⁺Thy1.2⁺CD4⁺ST2⁺) and ILC2s (CD45⁺Thy1.2⁺CD4⁻ST2⁺) were sorted using FACS Aria. 10,000 CD4 T_H2 cells and ILC2s were activated with PMA (100 ng/ml) and ionomycin (0.5 μg/ml) in a rounded 96-well plate for 4 h in the presence of Monesin and were then subjected to flow cytometry analyses for intracellular cytokines levels. The quantification of the mIL-5 cytokine in BAL supernatants of naïve and A.A.-treated mice were also performed by the Laboratory for Immunogenomics (RIKEN, IMS) via Luminex analysis.

OVA-induced airway inflammation model

8–12-wk-old male and female mice were first immunized with intraperitoneal administration of 50 μg of OVA (in 100 μl PBS) + 100 μl of Alum (Imject Alum Adjuvant, Thermo Fisher Scientific), on Day 0 and Day 13. On days 27, 28, and 29, the mice were anesthetized by isoflurane inhalation, followed by intranasal injection of 50 μg of OVA (in 40 μl PBS). After 24 h post final challenge, BAL and lungs of naïve and OVA-challenged mice were processed and analysed using the same methods for A.A-induced airway inflammation model.

In vitro activation of ILC2s

Lung cell suspensions from mice were prepared as outlined above. After Percoll gradient centrifugation, 5,000 ILC2s (CD45⁺Lin⁻CD3ε⁻Thy1.2⁺) were sorted into each well of 96 well-plate. Sorted ILC2s were cultured in 100 μl of complete medium (KOHJIN BIO DMEM-H [16003016] + 10% FBS) in the presence of mIL-33 (10 ng/ml), mIL-2 (10 ng/ml) and mIL-7 (10 ng/ml) for 4 d.

Statistical analysis

Student T tests and two-way ANOVA with post hoc Bonferroni tests were performed using GraphPad Prism software: ***P < 0.001, **P < 0.01, and *P < 0.05.