Fig 2: ILC2 and EOS interactions with ECs, DCs, and monocytes. A) Immunoblot detected ICAM-1 and VCAM-1 expression from aortic ECs pretreated with or without EOS lysates from WT mice or ILC2 lysates from WT or Il5-/- mice. B/C) Mouse aortic ring assay representative images and quantification. bFGF was used as positive controls. Scale: 400 µm. D) FACS analysis of bone-marrow-derived DCs cultured with or without EOS lysates and ILC2 lysates from WT mice. FACS analysis of CD11b+Ly6Chi monocytes after bone-marrow-derived monocytes were treated with E) LPS and INF-?, and F) CD11b+Ly6Clo monocytes after cells were treated with IL4, with and without EOS lysates and ILC2 lysates from WT mice. Representative images in panels (A) and (D) are shown to the left. Data are mean±SEM. n = 3–7 per group. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA test.

Fig 3: Brd4‐BD2 binds to acetylated‐EED to repress Foxp3 and Fbxw7 in Th2 cells ChIP‐PCR of the binding of Brd4 and Re‐ChIP‐PCR of YY1 and Gata3 on the gene loci of Il4, Foxp3, and Fbxw7 in mouse Th2 cells.ChIP‐qPCR analysis of the binding of G9a on the gene loci of Foxp3 and Fbxw7 in mouse Th2 cells treat with or without JQ1 (250 nM).Th2 cells were differentiated for 6 days, treated with JQ1 (250 nM) on Day 0. Lysates are immunoprecipitated with BRD4 followed by Western blotting analysis of BRD4, EED, and G9a.RNA‐seq data analysis showing expression of PRC2 key genes in mouse Th2 cells treated with or without JQ1 (250 nM). Data are representative of two biological replicates.HEK293T cells are transfected with Flag‐Brd4 and HA‐EED (left), HA‐Ezh2 (middle), or HA‐Suz12 (right) plasmids. Lysates are immunoprecipitated with Flag‐tagged Brd4, treated in vitro with PBS and Benzonase, followed by Western blotting of HA to detect HA‐EED, HA‐Ezh2, and HA‐Suz12.HEK293T cells are transfected with Flag‐Brd4 and HA‐EED plasmids. Lysates are immunoprecipitated with HA, followed by liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis. MS/MS spectrum for the identified acetylated peptides containing Lysine acetylation at position K19 and K211. Peak heights show the relative abundance of the corresponding fragmentation ions. The peptide sequences are shown at the top of the corresponding MS/MS spectrum with acetylated lysine residues highlighted in red. The identified fragmentation y (red color) and b (blue color) ions are indicated.Th2 cells were differentiated for 3 days and then treated with DMSO and JQ1 (250 nM) for 6, 24 and 48 h, followed by ChIP‐qPCR analysis of binding of Brd4 and EED on Foxp3 and Fbwx7. Data information: Mouse naïve CD4+ T cells were cultured in Th2 polarization condition and treated with or without inhibitors on Day 0 and were differentiated for 6 days before analysis, unless otherwise specified. All Western blotting data are representative of three independent experiments. All ChIP‐qPCR data represent mean ± SD and are representative of three independent experiments. Data are analyzed by Paired t test. *P < 0.05; **P < 0.01; and ***P < 0.001. Source data are available online for this figure.

Fig 4: Foxp3 inhibits Gata3 gene transcription while Fbxw7 promotes Gata3 protein degradation ATAC‐seq tracks of accessible cis‐regulatory regions of Foxp3, Fbxw7, Gata3, and Il4‐13 in mouse naïve, Th1, Th2, Th17, and Treg cells.qPCR analysis of Il4, Il5, Il13, Gata3, Foxp3, and Fbxw7 in mouse Th2 cells infected with sh‐Ctrl, sh‐Foxp3, and sh‐Fbxw7 lentivirus.Immunoprecipitation of Flag‐tagged Fbxw7 with anti‐Flag affinity gel, followed by Western blotting of Myc to detect Myc‐Gata3 in HEK293T cells transfected with Myc‐Gata3 and/or Flag‐Fbxw7 plasmids.Flow cytometric (left) and statistical analysis (right) of IL‐4 and Gata3 in mouse Th2 cells infected with sh‐ctrl and sh‐Fbxw7.Flow cytometric analysis of IL‐4 and Gata3 in mouse naïve CD4+ T cells cultured in Th2 polarization were treated with JQ1 (500 nM) at Day 0, and then treated with DMSO or MG132 (20 μM) for 6 h before harvest.Western blotting of Gata3 in mouse Th2 cells treated with or without JQ1 (500 nM), and then treated with cycloheximide (CHX; 25 μg/ml) for 0 h, 0.5 h, 1 h, and 2 h before harvest to stop protein synthesis.Co‐IP to immunoprecipitated Gata3, followed by Western blotting of ubiquitin in mouse naïve CD4+ T cells cultured in Th2 polarization treated with JQ1 (500 nM) at Day 0, and then treated with DMSO or MG132 (20 μM) for 6 h before harvest. Data information: Mouse naïve CD4+ T cells were cultured in Th2 polarization condition and treated with or without inhibitors on Day 0 and were differentiated for 6 days before analysis, unless otherwise specified. All Western blotting data are representative of three independent experiments. All data represent mean ± SD and average of three independent experiments. Data are analyzed by Paired t test. *P < 0.05; **P < 0.01; and ***P < 0.001. Source data are available online for this figure.

Fig 5: Brd4 through its BD2 domain regulates transcription of Th2‐key genes and ‐negative regulators Flow cytometric (left) and statistical analysis (right) of IL‐4 and IL‐17A in mouse Th2 and Th17 cells treated with BRD4 BD2‐selective inhibitor ABBV‐744 (1 μM) and BD1‐selective inhibitor MS611 (2 μM), respectively.qPCR analysis of Il4, Il5, Gata3 in mouse Th2 cells treated with ABBV‐744 at 500 nM and 1 μM.Western blotting of Gata3 in mouse Th2 cells treated with or without JQ1.Western blotting of Gata3 of mouse Th2 cells treated with ABBV‐744 (500 nM) and MG132 (20 μM).qPCR analysis of Foxp3 in mouse Th2 cells treated with ABBV‐744 at 500 nM and 1 μM.qPCR analysis of Fbxw7 in mouse Th2 cells treated with ABBV‐744 at 500 nM and 1 μM.Schematic diagram illustrating BRD4 functions in control of transcriptional activation and repression of key genes in chromatin that functionally cooperate with each other to regulate Th2 cell program. Histone lysine acetylation and methylation are depicted with color‐coded flags in green or red, respectively. Data information: Mouse naïve CD4+ T cells were cultured in Th2 polarization condition and treated with or without inhibitors on Day 0 and were differentiated for 6 days before analysis, unless otherwise specified. All data represent mean ± SD and are representative of three independent experiments. Data are analyzed by Paired t test. *P < 0.05; **P < 0.01; and ***P < 0.001. Source data are available online for this figure.