Fig 2: Oct-1/2 bind to the Il4 promoter and DHSs within the Th2 LCR.Binding of Oct-1/2 to the Il4 promoter (IL4P) and RHS5, RHS6, and RHS7 within the Th2 LCR was examined by a chromatin immunoprecipitation assay in Th1 and Th2 cells. Data are representative of three independent experiments with similar results. Statistical differences between groups were analyzed by Student’s t-test (*: P < 0.05).

Fig 3: Oct-1 induces Th2 cytokine expression.(A) Naïve CD4 T cells were activated, transduced with Oct1-MIEG3, Oct2-MIEG3, or control (empty) MIEG3 retroviral vector, and differentiated into Th2 cells. Cytokine expression was measured by intracellular cytokine staining. The plots show the percentages of differentiated cells expressing a particular cytokine. Data are representative of three independent experiments with similar results. (B) The results in (A) were shown as bar graphs. Statistical differences between groups were analyzed by Student’s t-test (*: P < 0.05). (C) Expression levels of Th2 cytokine mRNA. Activated T cells were transduced with Oct-1-MIEG3 or control MIEG3 vector as in (A). GFP+ cells were sorted from samples used in (A) and re-stimulated with anti-CD3 antibody for 24 h. The mRNA expression levels for selected Th2 cytokines were measured by quantitative PCR. Statistical differences between groups were analyzed by Student’s t-test (*: P < 0.05). (D) The effect of Oct1 siRNA on the expression of the Il4 gene. EL4 cells were transfected with control or Oct1 siRNA. The mRNA expression levels for the Il4 gene was measured by quantitative RT-PCR. Bars indicate mean ± SD (n = 3). Statistical difference between groups was analyzed by Student’s t-test (*: P < 0.05). (E) Effect of Oct-1 overexpression on cytokine expression in Th1 cells. Naïve CD4 T cells were transduced with Oct1-MIEG3 or control MIEG3 retroviral vector as in (A) and differentiated into Th1 cells. Cytokine expression was measured by intracellular cytokine staining. The plots show the percentages of cells expressing a particular cytokine. Data are representative of three independent experiments with similar results. (F) IFN-?-positive cells in (D) were shown as a bar graph.

Fig 4: ADNP is associated with genes involved in lymphocyte differentiation(A) Average ChIP-seq signal over all ADNP peaks in Th2 cells. Data are representative of 3 biological replicates.(B) Pie chart displaying distribution of 7,291 ADNP peaks across genomic features. Promoter includes peaks between 1-kb upstream and 1-kb downstream of the transcription start site (TSS). Downstream includes peaks up to 1-kb downstream transcription termination site (TTS).(C) KEGG pathway analysis of the genes associated to ADNP peaks. All pathways shown were enriched (p < 0.05).(D) Venn diagram showing the overlap between ADNP ChIP-seq peaks in Th2 and naive T cells. Peak list was generated using two biological replicates.(E) KEGG pathway analysis of the genes associated with ADNP gained peaks (peaks present in Th2 cells but not in naive T cells). All pathways shown were enriched (p < 0.05).(F) Top two enriched motifs of ADNP peaks in naive T cells and Th2 cells. Enrichment was assessed using a one-sided cumulative binomial distribution in HOMER.(G) Venn diagram showing the overlap between ADNP and CTCF ChIP-seq peaks in Th2 cells. Peak list was generated using two biological replicates.(H) Representative binding profiles of ADNP in wild-type naive T cells and Cd4Cre Th2 cells and CTCF in Cd4Cre or Cd4CreAdnpfl/fl Th2 cells at the Runx3 locus. Data are representative of 3 biological replicates.(I) Average ChIP-seq signal over all CTCF peaks in Cd4Cre or Cd4CreAdnpfl/fl Th2 cells. Data are representative of 2 biological replicates.(J) Representative binding profiles of ADNP in wild-type naive T cells and Cd4Cre Th2 cells; and CTCF in Cd4Cre or Cd4CreAdnpfl/fl Th2 cells at the type 2 cytokine locus. Black arrows indicate CTCF-binding sites (CBSs). Data are representative of 3 biological replicates.(K) Predicted CTCF motifs within the ADNP ChIP-seq peaks at the Il4 and Il13 promoter regions (magenta and purple rectangles in J). Prediction was performed using Jaspar transcription factor database.See also Figure S4 and Tables S3 and S4.