Fig 2: Batf Is Required for Chromatin Looping at the Il21 Locus in Activated CD4+ T Cells.(A–C) Chromosome conformation capture (3C) assay performed on 24-h and 96-h WT Th0 and Th0+IL-6 cells (A–C) and 96-h Batf KO cell (B and C) shows relative crosslinking frequencies between selected anchor fragments for the Il21 locus (promoter, +28 and -41; A and B) and Il17a/f locus (Il17a conserved non-coding sequence +11; C) and EcoRI fragments containing the indicated regions. Crosslinking frequencies were corrected for differences in ligation and PCR efficiency using reference DNA and normalized to control interaction frequencies with primer pairs within Gapdh locus.(D–F) Naive WT and Batf KO cells were activated under Th0+IL-6 conditions. On day 2, cells were transduced with control retrovirus vector (Thy1.1) or retroviral vector expressing Ets1-Thy1.1. Thy1.1-positive cells sorted on day 4 were used to assess gene expression by RT-PCR (D), Ctcf binding by ChIP-qPCR (E), and chromatin looping between Il21 promoter and indicated regions by 3C assay (F).Data are means ± SEM of three to five independent experiments with one individual mouse per experiment. *p < 0.05 (Student’s t test or one-way ANOVA followed by Tukey’s test).

Fig 3: Ets1 and Batf Are Required for Modulating Ctcf Occupancy in the Genome of Activated CD4+ T Cells.(A) Motif enrichment analysis of Batf-dependent Ctcf peaks exclusive of Batf binding represented by scatterplot with cyan dots indicating top 10 motifs with lowest p value. Analyzed peaks were collected by integrating Ctcf ChIP-seq data from 96-h WT Th0 and Th0+IL-6 versus Batf KO Th0 and Th0+IL-6 with Batf ChIP-seq peaks (Figure 1F).(B–E) Ets1 ChIP-seq peaks from 96-h WT and Batf KO Th0 and Th0+IL-6 subjected to motif-enrichment analysis identified Batf and Ctcf motifs represented by scatterplot with red dots indicating top motifs with the lowest p value (B). Ctcf, Batf, and Ets1 coincident and independent binding derived from Batf ChIP-seq (Figure 1F), Ctcf ChIP-seq (Figure 4D) and Ets1 ChIP-seq data from 96-h WT and Batf KO Th0 and Th0+IL-6 (as in B) (C). Scatterplots show normalized Ets1 ChIPseq tag density with numbers of differential peaks (fold change > 2; FDR < 0.05) compared between WT and Batf KO cells (D). Batf and Ets1 ChIP-seq peaks from WT and Batf KO cells integrated with enriched Ctcf peaks in WT compared with Batf KO (DCtcf WT versus Batf KO) in Th0+IL-6 cells identify Ctcf sites that are cobound with Ets1 and Batf or with Ets1 or Batf alone. Heatmap density shows normalized Batf, Ctcf, and Ets1 ChIP-seq signals compared between WT and Batf KO in Th0+IL-6 cells centered ± 1 kb of the indicated clusters. Percentages of ?Ctcf (WT versus Batf KO) sites associated with Batf and Ets1 sites are shown (E).(F) Ets1 expression in WT and Batf KO Th0 and Th0+IL-6 cells 96-h after stimulation assessed by RT-PCR. Values were normalized to expression in WT+IL-6 cells to calculate relative quantification.(G) Comparison of Batf- and Ets1-dependent Ctcf recruitment derived from Ctcf ChIP-seq peaks from Ets1cKO Th0 and Th0+IL-6 cells compared with WT and Batf KO ChIP-seq regions. Heatmap density shows normalized Ctcf ChIP-seq signals center ± 1 kb of clusters corresponding to Ctcf binding along with co-bound Ets1, Batf, or both. Percentages of Ctcf sites associated with Batf and Ets1 sites are shown.(H) Naive WT and Ets1 cKO CD4+CD62hi T cells were activated with anti-CD3 and anti-CD28 ± IL-6 for 96 h; total RNA was extracted, and gene expression was assessed by RT-PCR. WT cells were used as controls to calculate relative quantification.(I) Ctcf, Med 12, and Ets1 ChIP-seq data from WT, Batf KO, and Ets1 cKO Th0 and Th0+IL-6 and STAT3 ChIP-seq data from WT Th0 and Th0+IL-6 aligned to the extended Il21 locus. Blue shading highlights reduced binding in Batf KO and Ets cKO cells compared with WT cells. Data for STAT3 ChIP--seq are from GSE:65621.Data are means ± SEM of three to five independent experiments with one individual mouse per experiment (F and H), or representative of two independent with similar results (A–E, G, and I). RQ, relative quantification. *p < 0.05 (one-way ANOVA followed by Tukey’s test).

Fig 4: Batf Is Required for Global Chromatin Interaction in Activated CD4+ T Cells.(A) Circos plots of open chromatin, transcription factor occupancy, and intra-chromosomal interactions in the extended Il21 locus in WT (left) and Batf KO (right) Th0+IL-6 cells; 24-h WT and Batf KO Th0+IL-6 ATAC-seq data are displayed in the largest inner rings (black), and Batf (blue), Ctcf (green), and Ets1 (red) occupancy from 96-h WT and Batf KO Th0+IL-6 ChIP-seq data are shown in sequentially smaller inner rings. Significant (p < 0.005, resolution 10 kb) intra-chromosomal interactions identified by Hi-C analysis of 96-h WT and Batf KO Th0+IL-6 are shown by gray connecting lines. Black lines (left) indicate interactions that are lost in Batf KO cells. Red arrows indicate reduced Batf, Ets1, and Ctcf binding in Batf KO cells compared with WT cells.(B) Boxplots show the normalized intra- (top, 10 kb resolution) or inter- (bottom, 100 kb resolution) acting frequencies of chromosomal connections across the entire genome or regions containing Batf target genes in 96-h WT and Batf KO Th0 and Th0+IL-6 cells.(C) Principal-component 1 (PC1) from Hi-C data identified permissive chromatin across chromosome 3 (chr3; red track) aligned with ATAC-seq data (A). Matrix illustrates Pearson’s correlation coefficient of intra-chromosomal interactions in chromosome 3 normalized to observed interaction frequency to expected interaction frequency at 100 kb resolution. Blue, lower than expected (inactive); red, higher than expected (active).(D) Principal-component analysis of Hi-C data shows inter experimental correlation (different correlation values) calculated by comparing the two interaction profiles at each locus with PC1 values, defined at 50-kb intervals of chr3 for WT and Batf KO Th0+IL-6 cells. The different correlation value is high (close to 1) if the locus is likely to interact with similar regions in both samples and is low if the locus interacts with different regions.(E) Enrichment of interactions at accessible chromatin (50 kb resolution) in WT (top) and Batf KO (bottom) Th0+IL-6 cells using Structured Interaction Matrix Analysis (SIMA). Colors (red/blue) indicate association calculated as the log ratio of observed frequency to expected frequency (obs/exp).Data are representative of two independent experiments with similar results. *p < 0.05 (Mann-Whitney test).

Fig 5: Irf4 Is Dispensable for Batf-Regulated Chromatin Accessibility in CD4+ T Cells.(A) ATAC-seq analysis of 24-h activated WT andIrf4 conditional KO (cKO) Th0 and Th0+IL-6 cells. Scatterplots of normalized ATAC-seq tag density compare chromatin accessibility (fold change > 2; FDR < 0.05) between 24-h activated WT and Irf4 cKO Th0 and Th0+IL-6 cells.(B) Irf4 ChIP-seq peaks from WT cells along with Batf ChIP-seq peaks (Figures 1F and 1G) integrated with chromatin accessibility (Figure 1F) identify unique Batf and Irf4 sites along with sites of Batf and Irf4 co-binding that are associated with closed and open chromatin regions. Heatmap density shows normalized Batf and Irf4 ChIP-seq signals in WT Th0 and Th0+IL-6 cells centered ± 1,000 bp of the indicated clusters. Percentages of Batf and Irf4 sites associated with closed and open chromatin are indicated.(C) Heatmap density shows normalized ATAC-seq signals centered ± 500 bp of unique Batf and Irf4 sites along with co-bound Batf and Irf4 sites with percentages of sites compared between WT and Irf4-deficient cells in Th0 and Th0+IL-6 conditions.Data are representative of two independent experiments with similar results