Fig 2: 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.

Fig 3: Absence of ADNP at the Il13 promoter reduces local H3K27 acetylation and DNA accessibility(A) Representative ATAC-seq and H3K27ac ChIP-seq tracks from Th2 cells from Cd4Cre or Cd4creAdnpfl/fl mice in the type 2 cytokine locus. Data are representative of 2 biological replicates.(B) Detection of p300 protein (218 kDa) in immunocomplex generated with Th2 cell nuclear lysate co-immunoprecipitated with anti-ADNP antibody. INP, input nuclear extract; SN, supernatant; IP, immunoprecipitation elution. Data are representative of 2 independent experiments. IB refers to immunoblotting antibody.(C) Average ChIP-seq signal over all H3K27ac peaks or over ADNP-CHD4-BRG1 peaks in Cd4Cre or Cd4CreAdnpfl/fl Th2 cells. Data are representative of 3 biological replicates.(D) Average ATAC-seq signal over all H3K27ac peaks or over H3K27ac peaks that were reduced in Cd4CreAdnpfl/fl Th2 cells. Data are representative of 2 biological replicates.(E) KEGG pathway analysis of genes associated to the genomic regions where H23K27ac was reduced in Cd4CreAdnpfl/fl Th2 cells. All shown pathways were enriched (p < 0.05).(F) Representative binding profiles of ADNP, CHD4, BRG1, and H3K27ac in Th2 cells of Cd4Cre or Cd4CreAdnpfl/fl mice at the Maf and Il4ra loci. Data are representative of 2 biological replicates.See also Figure S7 and Table S6.

Fig 4: Single-cell gene expression analysis confirms the importance of ADNP for IL-13 expression(A) UMAP plot of single-cell gene expression analysis of naive CD4+CD44-CD62L+CD25- T cells and CD4+CD44+CD62L-ST2+ effector T cells purified from the lungs of papain-challenged Cd4Cre and Cd4CreAdnpfl/fl mice.(B) UMAP plot with expression (log2 expression) of indicated genes per individual cell.(C and D) Volcano plot comparing genes from naive T cells (cluster 1) (C) and Th2 cells (cluster 2) (D) from Cd4Cre and Cd4CreAdnpfl/fl (KO) mice.(E) UMAP plot with expression (log2 expression) of Il13 per individual cell.(F) Violin plots with expression (log2 expression) of indicated genes. Rectangle, solid line, and dashed line represent the interquartile range, the median and the mean, respectively.(G) Heatmap showing the expression of indicated genes in cells from cluster 2 from Cd4Cre (left) and Cd4CreAdnpfl/fl (right) mice. Columns represent cluster 2 individual cells and rows represent the different genes.(H) UMAP plot showing cluster 2. Cells are colored by their cytokine expression pattern as shown, with number of cells indicated in parentheses.See also Figure S3.