Fig 2: Results of bulk RNA-seq of CD4 T cells.(A) Spleen CD4 T cells from Cbx7+/+ and Cbx7−/− mice were stimulated with anti-CD3/28 antibodies for 6 hours. RNA was isolated and processed for bulk RNA-seq. The volcano plot shows down- and up-regulated genes in CD4 T cells from CBX7 KO and WT mice. (B) A heatmap of top 50 DEGs (differentially expressed genes) of stimulated CD4 T cells from WT and CBX7 KO mice. (C to E) Validation of the RNA-seq data for select type-2 genes by qPCR using RNA from stimulated CD4 T cells (n = 6). (F to H) Effect of the CBX7 inhibitor MS37452 on expression of mRNA for ERDR1, SYNJ2, and ACSS2 in PBMCs from asthmatic donors (n = 10). (I and J) KEGG pathway enrichment analysis of differentially expressed genes using Enrichr. The bar charts show the most relevant and significantly enriched terms. The x axis represents the −log10 of the enrichment P value. The y axis represents the enriched terms in KEGG databases. KEGG pathway enrichment analysis of differentially expressed genes were performed using Enrichr under default parameters (49).

Fig 3: The CBX7 inhibitor MS37452 abrogates allergic asthma in an ILC-dependent mouse model.A schematic diagram of allergen exposure to C57BL/6J WT. (A) WT B6 mice were pretreated with the Veh (DMSO) or the CBX7-inh (319 μg per dose) and 1 hour later, sensitized intranasally (IN) with the Alternaria (Alt) allergen extract (10 μg/ml) for 5 consecutive days. Treatment with the CBX7-inh reduced AHR (B), lung inflammation and mucus production (C and D), and the numbers of lung infiltrating eosinophils (E to G) and IL-5 and IL-13 secreting ILC2s (H to J). (K) A schematic diagram of allergen exposure to Rag1−/− mice. The CBX7-inh reduced AHR (L), lung infiltrating eosinophils (M), the frequency of IL-5+ and IL-13+ ILCs in the lung (N to P), and the levels of IL-4, IL-5, and IL-13 in BAL (Q to S). D, day; BM, basement membrane; RL, lung resistance; Sal, saline.

Fig 4: CBX7 binds to cytokine gene promoters and interacts with transcriptional coactivators.(A and B) Blood CD4 T cells were stimulated with anti-CD3/28 antibodies for 72 hours. ChIP-grade anti-CBX7 and control rabbit IgG were used to immunoprecipitate the bound chromatin. DNA was used for qPCR using primers for the proximal promoter, the enhancer region, and the conserved CNSs for type-2 genes. Fold enrichment is shown as the percentage of input (1:100 dilution), *P ≤ 0.05 (n = 3). (B) Primers from the promoter of the p16 gene were used as a positive control. (C to E) Distribution of various histone marks on cytokine genes and regulatory regions. ChIP-grade H3K27me3, H3K4me3, H3K27ac, or con-IgG were used for ChIP of CD3/28-stimulated CD4 T cells (n = 3). (F) Interaction of CBX7 with P-300, CK2-α, and Pol-II. Cell lysate from stimulated CD4 T cells was immunoprecipitated with an anti-CBX7 or control IgG (con) antibodies and Western blotted for P-300, Pol-II, and CK2-α (n = 3). IP, immunoprecipitation; LE, long exposure; SE, short exposure; WCL, whole cell lysate. (G and H) Cell lysate from stimulated CD4 T cells was immunoprecipitated with an anti-P300 antibody, Western blotted for CBX7 and P-300, and reprobed for acetyl lysine. (I) Primary HBECs were cultured with and without EGF (10 ng/ml) for 48 hours. The cell lysate was immunoprecipitated with the anti-CBX7 antibody and Western blotted for Pol-II, CK2-α, and MEK2. n = 3. IgG-H, IgG heavy chain; IgG-L, IgG light chain.

Fig 5: CBX7 regulates cytokine production by lymphoid cells.(A) The expression of mRNA for the CBX7 (1–8) family proteins in the lung of allergen (Alternaria) or saline-sensitized mouse models of asthma (n = 4 mice per group). (B) CBX7 mRNA expression in PBMCs analyzed by reverse transcription quantitative real-time PCR. (C) Western blots show CBX7 expression in CD4 T cells after 72 hours of anti-CD3/28 antibody stimulation. The numbers below the blot indicate CBX7 levels normalized to actin (n = 3). (D and E) Effect of the CBX7 inhibitor (CBX7-inh) MS37452 on IL-4+ ILCs. Blood Lin− cells were cultured with IL-2/IL-33 (20 ng/ml each) and increasing doses of the CBX7-inh for 72 hours. IL-4+ ILCs were measured by FCM (N = 4). (F to J) PBMCs from asthmatics were cultured either with DMSO (Veh) or the CBX7-inh (100 μM) for 72 hours. Representative flow plots and graphs show the expression of IL-4 and IL-5 in Lin− cells and Lin+ cells (n = 16). (K and L) IL-5 and IL-13 protein levels in culture supernatants from the experiments for FCM [(H) to (L)]. (M and N) BAL cells were cultured with the CBX7-inh (100 μM) for 72 hours. IL-5 expression by Lin−IL-7Rα+CRTH2+ ILC2s was measured by FCM (n = 8). (O) CD3/28–stimulated PBMCs were transduced with a bicistronic GFP expressing pZMP lentiviral vector containing nontargeting (con) shRNA or CBX7 targeting shRNA—sh1, sh2, and sh3—for 72 hours and analyzed by Western blotting. Numbers below the CBX7 blot indicate the relative CBX7 levels compared to con-sh, which was normalized to β-actin (n = 3). (P and Q) Flow plots and bar graphs show the effect of CBX7 knockdown with CBX7-sh2 on IL-4+ CD4 T cells (n = 3). P values are shown above the graphs. The Wilcoxon signed-rank test was used to calculate the P value for data presented in (G) to (N). FSW, forward scatter width; CRTH2, chemoattractant receptor homologous molecule expressed on T helper type 2 cells.