Extended Data Fig. 7: Altered chromatin accessibility at regulated target genes of E2A, Ebf1, Pax5, and Ikaros.

a, Quality control of the DNA-binding data, generated by CUT&RUN²⁹ with an anti-V5 antibody in untreated pro-B cells of the 4 different TF genotypes. The indicated consensus recognition motifs of E2A (Tcf3), Ikaros (Ikzf1), and Ebf1 were identified with the indicated E values at the respective CUT&RUN peaks by de novo motif discovery⁷⁶. The more complex Pax5 recognition motif²² was identified at the center of the respective CUT&RUN peaks with CentriMo⁷⁷. Supplementary Table 3 contains additional results, including differential motif analyses based on chromatin accessibility changes. b, Open chromatin regions were determined by ATAC-seq analysis³⁰ of pro-B and pre-B cells expressing the indicated Aid-tagged TFs. ATAC peaks without TF binding are shown in black, while ATAC peaks with TF binding are shown in grey together with their percentage relative to the total ATAC peaks. c, Numbers of differential ATAC peaks (>4-fold) with or without TF binding are shown in dark or light colors, respectively. The percentages of TF-bound, differential ATAC peaks relative to all differential ATAC peaks (>4-fold) are indicated. d, Volcano plots highlighting the TF-bound peaks with a > 2-fold or 4-fold decrease (green) or increase (orange) of ATAC-seq signals in pro-B cells following TF degradation. The bound ATAC peaks with a > 4-fold decrease correspond to 11% (239/2,124), 28% (911/3,271), or 24% (635/2,613) of all E2A-, Ebf1-, or Pax5-dependent differential ATAC peaks, respectively. e, Relationship between differential ATAC peaks (>2-fold) and gene transcription in pro-B cells. Densities of transcription fold changes are shown for genes with TF-bound, differential ATAC peaks exhibiting decreased (green) or increased (orange) signals. Dashed lines indicate genes with TF-bound, differential ATAC peaks in their promoter region (−2.5 kb to +1 kb). As a control, densities of TF-bound, expressed genes (>10 TPM) with unchanged ATAC peak signals ( < 1.1-fold) are shown in grey. Blue and red shading denotes > 2-fold activated and repressed target genes, respectively. f, Chromatin accessibility changes and binding of the indicated TFs at two putative enhancers of the non-regulated gene Bmf. Intronic transcript levels (above) and ATAC peaks (below) were determined in small pre-B cells before (–) and after (+) 5-Ph-IAA treatment, while TF binding was determined in untreated control pre-B cells. g, Low frequency of non-regulated genes containing TF-bound, differential ATAC peaks. Non-regulated TF-bound genes were defined by a < 1.1-fold transcriptional change and a transcript value of > 10 TPM. The fraction (black bar) of genes containing differential ATAC peaks (adjusted P value of < 0.1 and a basemean value of > 100) is indicated. h, Activation of the Xrcc6, Esp8, and Egfl6 genes by E2A, Ebf1, and Pax5, respectively. The transcript abundance was determined before (–) and after (+) degradation of the respective TF in small pre-B cells. The indicated fold changes refer to DESeq2-derived fold changes of normalized transcription values. i, Density plot of fold changes of ATAC-seq signals at > 2-fold activated (blue) and repressed (red) target genes of the indicated TFs in pro-B cells. Only the TF-bound ATAC peak with the most significant fold change per gene was selected. ATAC peak signals associated with non-regulated genes ( < 1.1-fold) are shown as control (grey). Mean TPM values with SEM (f,h) and fold changes (b-h) are based on two total RNA-seq, ATAC-seq, or CUT&RUN experiments. Each vertical dash corresponds to one gene (e) or one ATAC-peak per gene (i).