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 stimulates transactivation activity of the Il4 promoter through RHS5.(A) EL4 cells were transfected with expression vectors (Oct1-CMV, Oct2-CMV, GATA-3-CMV, or control CMV) and the RHS5-IL4 reporter construct by electroporation. Cells were allowed to rest for 18 h, followed by treatment with PMA (50 ng/ml) and ionomycin (1 µM) for 4 h. Luciferase activity was then measured. Data are representative of three independent experiments with similar results (NT = no treatment). (B) Mutation sites of Oct-1-binding sites within Il4 promoter and RHS5. Oct-1-binding sites, close to GATA-3-binding site, within Il4 promoter were deleted as indicated (upper), or Oct-1 binding site adjacent to GATA3 binding site (Fig 2E) within RHS5 was mutated as indicated (lower). (C) RHS5(WT)-IL4P(WT) reporter construct or RHS5(mtOct1)-IL4P(WT) construct (RHS5-IL4P reporter construct bearing mutation at Oct-1 binding site within RHS5) were transfected into EL4 cells with expression vectors (Oct1-CMV, GATA-3-CMV, or control CMV) by electroporation. Cells were allowed to rest for 18 h, followed by treatment with PMA (50 ng/ml) and ionomycin (1µM) for 4 h. Luciferase activity was then measured. (D) RHS5(WT)-IL4P(?Oct1) (RHS5-IL4P construct in which Oct1 binding sites were deleted within the Il4 promoter) or RHS5(mtOct1)-IL4P(?Oct1) (RHS5-IL4 reporter construct bearing both mutation at Oct-1 binding site within RHS5 and deletion of Oct-1 binding sites within the Il4 promoter) were transfected into EL4 cells with expression vectors (Oct1-CMV, GATA-3-CMV, or control CMV) by electroporation. Luciferase activity was measured as (C). Data are representative of three independent experiments with similar results (NT = no treatment). Bars are shown to indicate mean ± SD (n = 3). Statistical difference between groups was analyzed by Student’s t-test (*: P < 0.05).

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.