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Transcriptional function of E2A, Ebf1, Pax5, Ikaros and Aiolos analyzed by in vivo acute protein degradation in early B cell development

Nature Immunology volume 25, pages 1663–1677 (2024)Cite this article

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An Author Correction to this article was published on 21 March 2025

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Abstracts

Early B cell lymphopoiesis depends on E2A, Ebf1, Pax5 and Ikaros family members. In the present study, we used acute protein degradation in mice to identify direct target genes of these transcription factors in pro-B, small pre-B and immature B cells. E2A, Ebf1 and Pax5 predominantly function as transcriptional activators by inducing open chromatin at their target genes, have largely unique functions and are essential for early B cell maintenance. Ikaros and Aiolos act as dedicated repressors to cooperatively control early B cell development. The surrogate light-chain genes Igll1 and Vpreb1 are directly activated by Ebf1 and Pax5 in pro-B cells and directly repressed by Ikaros and Aiolos in small pre-B cells. Pax5 and E2A contribute to V(D)J recombination by activating Rag1, Rag2, Dntt, Irf4 and Irf8. Similar to Pax5, Ebf1 also represses the cohesin-release factor gene Wapl to mediate prolonged loop extrusion across the Igh locus. In summary, in vivo protein degradation has provided unprecedented insight into the control of early B cell lymphopoiesis by five transcription factors.

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Fig. 1: Characterization and expression of Aid-tagged TF alleles.
Fig. 2: Impaired B cell development on prolonged degradation of Aid-tagged TFs.
Fig. 3: Identification of direct target genes by acute TF degradation.
Fig. 4: Unique and combinatorial target gene regulation by E2A, Ebf1, Pax5, Ikaros and Aiolos in early B cell development.
Fig. 5: Altered chromatin accessibility at regulated target genes of E2A, Ebf1, Pax5 and Ikaros.
Fig. 6: Direct control of transcriptional regulator genes by E2A, Ebf1, Pax5 and Ikaros in pro-B cells.
Fig. 7: Direct regulation of genes involved in pre-BCR expression and V(D)J recombination.
Fig. 8: Role of Ebf1 in the transcriptional repression of the Wapl gene.

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Data availability

The total RNA-seq, ATAC-seq and CUT&RUN-seq data reported in the present study (Supplementary Table 6) are available at the Gene Expression Omnibus (GEO) repository under accession no. GSE260523. Source data are provided with this paper.

Code availability

No original code is reported in this manuscript.

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Acknowledgements

We thank F. Stewart (Technical University, Dresden) for providing the Rosa26Tir1/+ mouse, C. Theussl’s team for generating gene-modified mice, K. Aumayr’s team for flow-cytometric sorting and A. Sommer’s team at the Vienna BioCenter Core Facilities for Illumina sequencing. This research was funded by Boehringer Ingelheim, the Austrian Research Promotion Agency (grant no. FFG-878286), the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant no. 740349 to M.B.) and in part by the Austrian Science Fund (grant no. P34372 to T.A.S.).

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Author notes

  1. These authors contributed equally: Anna S. Fedl, Hiromi Tagoh, Sarah Gruenbacher.

Authors and Affiliations

  1. Research Institute of Molecular Pathology, Vienna BioCenter, Vienna, Austria

    Anna S. Fedl, Hiromi Tagoh, Sarah Gruenbacher, Qiong Sun, Robyn L. Schenk, Kimon Froussios, Markus Jaritz, Meinrad Busslinger & Tanja A. Schwickert

  2. Vienna BioCenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, Vienna, Austria

    Anna S. Fedl & Sarah Gruenbacher

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Contributions

T.A.S., A.S.F. and S.G. performed the total RNA-seq experiments and phenotyping analyses with some initial help from R.L.S. H.T. performed the ATAC-seq and CUT&RUN experiments. Q.S. generated the Rosa26Tir1-F74G allele and all Aid-tagged TF alleles. M.J. bioinformatically processed and analyzed the total RNA-seq, ATAC-seq and CUT&RUN-seq data with initial help from K.F. T.A.S. and A.S.F analyzed the bioinformatic data. M.B. and T.A.S. planned the project, designed the experiments and wrote the manuscript with the help of A.S.F. and H.T.

Corresponding authors

Correspondence to Meinrad Busslinger or Tanja A. Schwickert.

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Nature Immunology thanks Masaki Miyazaki and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: A. Dempsey, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Early B-cell development in Tcf3Aid/Aid, Ebf1Aid/Aid, Pax5Aid/Aid, Ikzf1Aid/Aid, and Ikzf3Aid/Aid mice.

a, The indicated C-terminal tag sequences, consisting of a glycine linker, a minimal auxin-inducible degron (Aid) sequence65 and the V5 epitope, were added in frame to the last amino acid of E2A, Pax5, Ikaros, or Aiolos. The stop codon is denoted by an asterisk. b, Schematic diagram describing the generation of the Ebf1Aid allele. The exon-intron structure of the 5′ region of the Ebf1 gene is shown together with the sequences encoding the V5 epitope, Aid sequence, glycine linker and the exon 1-encoded amino acid sequence inserted into the 5′ part of exon 2 of the Ebf1Aid allele (for DNA sequence see Supplementary Table 5). c, Analysis of early B-cell development in the bone marrow of Tcf3Aid/Aid, Ebf1Aid/Aid, Pax5Aid/Aid, Ikzf1Aid/Aid, Ikzf3Aid/Aid, and control (Ctrl) wild-type mice at the age of 5 weeks. The flow-cytometric analysis of the different mice was performed on the same day in one experiment. The percentage of cells is indicated next to the gate of the different B-cell types. One of three experiments is shown. Imm, immature; Rec, recirculating. d, Expression of the indicated Aid-tagged TFs in ALPs, BLPs, and pro-B cells, which was determined by flow-cytometric analysis of intracellular staining with an anti-V5 antibody. The histogram displays the protein levels of the different TFs in the three progenitors. The flow-cytometric definition of the different B-cell types (c,d) is also described in Methods.

Extended Data Fig. 2 B-cell developmental defect due to spontaneous protein degradation in vivo by the Rosa26Tir1 allele, but not by the Rosa26Tir1-F74G allele.

a, Impaired B-lymphopoiesis in Pax5Aid/Aid Rosa26Tir1/Tir1 mice in the absence of auxin (IAA). B-cell development in the bone marrow of 3-4-week-old Pax5Aid/Aid Rosa26Tir1/Tir1, Pax5Aid/Aid Rosa26Tir1-F74G/Tir1-F74G, and control Pax5+/+ mice was investigated by flow-cytometric analysis. The percentage of B-cell types is indicated next to the gates. Pax5 protein expression in pro-B cells was determined by intracellular anti-Pax5 staining. One of two independent experiments is shown. b, No effect of the Rosa26Tir1-F74G allele in the absence of 5-Ph-IAA treatment on B-cell development in Tcf3Aid/Aid, Ebf1Aid/Aid, Pax5Aid/Aid, Ikzf1Aid/Aid, Ikzf3Aid/Aid, and Ikzf1,3Aid/Aid mice expressing the Rosa26Tir1-F74G allele. Frequencies of total B, pro-B, pre-B, and immature B cells were analyzed by flow cytometry and presented as mean values with SEM (n = 32,24,13,22,20,7,4). Statistical data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test: *P < 0.05, **P < 0.01, ****P < 0.0001. Each dot corresponds to one mouse. c, Time course analysis of Ikaros degradation for up to 25 h upon 5-Ph-IAA injection into Ikzf1Aid/+ Rosa26Tir1-F74G/+ mice. Aid-V5-tagged Ikaros protein levels were determined by flow-cytometric analysis of intracellular anti-V5 straining. The kinetics of Ikaros degradation is shown as geometric mean fluorescence intensity (gMFI) of the anti-V5 staining in pro-B, small pre-B and immature B cells relative to the gMFI of the untreated control (left; n = 2). One of two independent 25-hour experiments is shown. The histogram displays the Ikaros protein levels in the three B-cell types at the indicated time points after 5-Ph-IAA injection (right). d, Flow-cytometric sorting of pro-B, small pre-B, and immature B cells from the bone marrow of 4-week-old Ebf1Aid/Aid Rosa26Tir1-F74G/Tir1-F74G mice (upper row). The different gates used for flow-cytometric sorting are indicated together the percentage of cells in the respective gates. The purity of the sorted pro-B cell population was determined by reanalysis (lower row).

Source data

Extended Data Fig. 3 Impaired B-cell development upon prolonged degradation of E2A, Ebf1, Pax5, and Ikaros.

Flow-cytometric analysis of bone marrow from 6–10-week-old wild-type (Ctrl), Tcf3Aid/Aid, Ebf1Aid/Aid, Pax5Aid/Aid, Ikzf1Aid/Aid, Ikzf3Aid/Aid, and Ikzf1,3Aid/Aid mice expressing the Rosa26Tir1-F74G allele, which were analyzed after three (a,g) or six (f) days, or 18 hours (b-e) upon daily 5-Ph-IAA injection. a,b, The frequencies of ALPs, BLPs (a) and total B cells (b, upper row) are indicated next to the respective gate. The frequency of the CLPs in total bone marrow cells is also shown in (a). One of two independent experiments is shown. Short-term BrdU incorporation (b, lower row) in the last 45 minutes of the 18-hour degradation of the indicated TFs was analyzed by flow cytometry with Vpreb1 [CD179a] staining. The Vpreb1 expression allowed to distinguish between Vpreb1hiB220+CD19+/intIgM–IgD– cells (pro-B and recently generated large pre-B cells), Vpreb1+/intB220+CD19+/intIgM–IgD– cells (large pre-B cells), and Vpreb1–B220+CD19+/intIgM–IgD– cells (small pre-B cells). c, Quantification of bone marrow (BM) and B cells after 18 hours of TF degradation relative to untreated mice of the same genotype (n = 6,3,4,3,4). The gray bar refers to the ratio of two untreated littermates. d, CD19 expression on B cells before and after 18-hours TF degradation (n = 5/4,6/5,4/4,2/4). e, BrdU incorporation in the B220+CD19+/intIgM–IgD– B-cell population comprising pro-B and pre-B cells, based on the flow cytometric data shown in b (bottom row). n = 4/4,5/5,4/4,3/3. f, Impaired B-cell development upon 6-day degradation of Ikaros, Aiolos, or Ikaros plus Aiolos in Ikzf1Aid/Aid, Ikzf3Aid/Aid, and Ikzf1,3Aid/Aid mice, homozygous for Rosa26Tir1-F74G. Bone marrow cells of treated and untreated control mice were analyzed by flow cytometry to determine the frequencies of total B, pro-B, large, small pre-B, and immature B cells (n = 3,3,3,3). g, Developmental arrest at an aberrant large pre-B cell stage upon 3-day degradation of Ikaros and Aiolos. Only IgμhiVpreb1hiKit– B cells were detected by intracellular (i.c.) staining after prolonged protein degradation, while pro-B (Igμ–Vpreb1hiKit+), large pre-B (IgμhiVpreb1hi-intKit–), and small pre-B (IgμintVpreb1–Kit–) cells were identified in untreated mice. Statistical data (c-f) are shown as mean values with SEM and were analyzed by two-way ANOVA with Dunnett’s (c) multiple comparison test and by one-way ANOVA with Šídák’s (d,e) or Dunnett’s (f) multiple comparison tests: **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot (c-f) corresponds to one mouse.

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Extended Data Fig. 4 Comparison of Ikaros-regulated genes identified by Ikaros degradation and conditional Ikzf1 mutagenesis in pro-B cells.

a, Flow-cytometric analysis of B-cell development in the bone marrow of 3–6-week-old mice of the indicated genotypes after short-term 5-Ph-IAA-induced degradation (6 h: E2A, Ebf1, Pax5; 8 h: Ikaros, Aiolos, and Ikaros plus Aiolos). Total B and pro-B cell frequencies in treated (+) mice were compared with those of untreated (–) mice, analyzed in Extended Data Fig. 2b. Mean values with SEM are shown (n = 24/27,13/8,22/14,20/11,7/4,4/4). B cell frequencies of each genotype were unaltered by short-term treatment, as shown by the unpaired two-tailed Mann-Whitney test. b,c, Protein expression of E2A, Ebf1, Pax5, or Ikaros target genes. b, Identification of the Cd36, Cd72, Irf4, and Ikzf3 genes as directly regulated genes of E2A, Ebf1, Pax5, and Ikaros in pre-B or pro-B cells, respectively, as determined by total RNA-seq analysis before (–) or after (+) acute TF degradation. c, Flow-cytometric analysis of cell surface protein (CD36, CD72) and TF (Irf4, Aiolos) expression in pre-B or pro-B cells before (0 h) or after acute (6 h or 8 h) or prolonged (24 h or 72 h) degradation. d, Analysis of Bcl-xL expression by intracellular staining before (black line) or after 18 hours of TF degradation (colored surface). e, The gMFI of Bcl-xL staining in the indicated B-cell types was determined before and after 9 (n = 2) or 18 (n = 4,5,4,3) hours of degradation, and is displayed as a ratio, which was analyzed by two-way ANOVA with Dunnett’s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). f, Identification of Ikaros-regulated genes by analyzing intronic (left) or exonic (right) reads upon Ikaros degradation. Volcano plots highlight the differentially transcribed genes identified by total RNA-seq of pro-B cells from Ikzf1Aid/Aid Rosa26Tir1-F74G/Tir1-F74G mice before or after 8-hour Ikaros degradation. Differentially regulated genes were identified as increased (repressed, red) or decreased (activated, blue), if their intronic or exonic transcript abundance exhibited a shrunken fold change of > 2, an adjusted P value of < 0.001 and a mean transcript value of > 10 TPM (intronic) or > 5 TPM (exonic) in experimental or control pro-B cells. Correlation between intronic and exonic transcript fold changes is shown below. g, Comparison of the gene expression differences observed upon acute Ikaros protein degradation or conditional Ikzf1 gene deletion in pro-B cells. The previously published analysis of mutant Cd79a-Cre Ikzf1fl/– and control Cd79a-Cre Ikzf1fl/+ pro-B cells6 identified regulated genes by mRNA-seq, if they passed the described selection criteria (e) with a shrunken fold-change of > 3. The correlation between the regulated genes identified by acute Ikaros protein degradation and conditional Ikzf1 gene deletion is shown below. The numbers (f,g) of identified regulated genes are shown. Fold changes (b,f,g) refer to DESeq2-derived fold changes of normalized transcription values. Mean TPM values with SEM (b) and fold changes (b,f,g) are based on two RNA-seq experiments per condition. Each dot corresponds to one mouse (a,e), one total RNA-seq experiment (b) or one gene (f,g).

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Extended Data Fig. 5 Identification of direct target genes of the different TFs by protein degradation in pro-B, small pre-B, and immature B cells.

a, Numbers of regulated genes identified by analyzing the nascent transcript data (generated for the different TFs and cell types by acute protein degradation; Fig. 3d) with different fold change parameter. Genes were identified as increased (repressed, red) or decreased (activated, blue), if they exhibited an adjusted P value of < 0.001 and a mean transcript value of > 10 TPM in the experimental or control B-cell types. In addition, genes were selected by fold change (FC) criteria of > 2, > 1.5 or none. b, Altered transcript levels in Ebf1 and Pax5 untreated pro-B cells (grey bars). The transcript abundance at the Gsn, Cd19, Ptpre, and Emb genes was determined as mean TPM value with SEM by total RNA-seq before (–) and after (+) degradation of E2A, Ebf1, Pax5 and Ikaros in B-cell subsets of Tcf3Aid/Aid, Ebf1Aid/Aid, Pax5Aid/Aid, and Ikzf1Aid/Aid mice homozygous for Rosa26Tir1-F74G. Red dashed horizontal lines indicate the TPM value of the Gsn, Cd19, Ptpre, and Emb transcripts in untreated E2A control pro-B cells. Red arrow indicated the decrease or increase of these transcripts in Pax5 control pro-B cells (Cd19, Ptpre) and Ebf1 control pro-B cells (Gsn, Emb). c, Systematic analysis of activated (blue) and repressed (red) genes (>1.5-fold) by comparing the distribution of transcription fold changes between untreated control pre-B cells of the indicated genotypes. As only few genes are deregulated upon Aiolos degradation (a), we used the untreated control small pre-B cells of Ikzf3Aid/Aid Rosa26Tir1-F74G/Tir1-F74G mice as a reference to calculate the corresponding shrunken log2 fold-changes of the untreated control cells of the 4 indicated genotypes. Each horizontal dash corresponds to one gene. The indicated fold-changes (b,c) refer to DESeq2-derived fold changes of normalized transcription values. Mean TPM values (b) and fold changes are based on two total RNA-seq experiments.

Extended Data Fig. 6 Overlap of the target gene signatures of different transcription factors in early B-cell development.

a, Overlap analysis of the gene lists consisting of the directly regulated target genes of the different TFs, identified by acute protein degradation in small pre-B cells. Horizontal bars in the UpSet plot indicate the total identified target genes of the different TFs, while vertical bars display the counts of unique or shared target genes across TFs. Combinatory categories with less than 10 genes are not displayed. The percentages refer to the unique target genes relative to the total target genes identified for each TF. The target genes analyzed were defined by a difference in intronic transcripts of a shrunken fold-change of > 1.5, an adjusted P value of < 0.001 and a mean transcript value of > 10 TPM in the 5-Ph-IAA-treated or control small pre-B cells. b, GSEA analysis as described in Fig. 4c except that an FDR threshold of < 0.01 was used. c, Gene set enrichment plots and corresponding normalized enrichment score (NES) displayed in (b) and Fig. 4c. As shown by this example, the E2A activated target gene set, which was identified by a shrunken fold-change of > 1.5 (Extended Data Fig. 5a) upon E2A degradation in pro-B cells, was compared with the shrunken fold change-ranked gene list (x-axis) determined by degradation of the indicated TF in the specified B cell type. The enrichment score (ES) is shown on the y-axis. Gray color indicates results obtained with an FDR of > 0.01.

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).

Extended Data Fig. 8 Direct regulation of transcriptional regulator genes by E2A, Ebf1, Pax5, and Ikaros in immature B cells.

a, Heatmaps of gene expression changes of transcriptional regulators that are controlled by E2A, Ebf1, Pax5, and Ikaros in immature B cells. The four groups of E2A-, Ebf1-, Pax5-, and Ikaros-regulated genes, which were identified by a > 1.5-fold difference in transcript abundance upon protein degradation in immature B cells, were each sorted according to their fold change in immature B cells (black boxes). b, Direct activation of the Zfpm1 (Fog1) gene by E2A and Ebf1 in small pre-B and immature B cells. The Zfpm1 transcript abundance was determined before (–) and after (+) 5-Ph-IAA-mediated degradation of E2A or Ebf1, respectively. The indicated fold changes refer to DESeq2-derived fold changes of normalized transcription values. Mean TPM values with SEM (b) and fold changes (a,b) are based on two total RNA-seq experiments.

Extended Data Fig. 9 Regulation of surrogate light-chain genes as well as Irf4, Bcl2l1, and Ikzf1 in early B-cell development.

a, Schematic diagram of the pro-B to pre-B cell transition, which is controlled by signaling of the pre-BCR consisting of the Igμ chain, the surrogate light chains Igll1 (λ5) and Vpreb and the signaling chains Igα and Igβ. Rag1, Rag2, and Dntt expression and the Irf4 and Irf8 upregulation during the large to small pre-B cell transition are shown. b, Schematic diagram of the locus containing Igll1 and Vpreb1. The intron sizes (blue) are indicated in base pairs (bp) and the distance between the Igll1 and Vpreb1 genes in kb. c, Regulation of Vpreb1, Vpreb2, and Vpreb3 in pro-B and small pre-B cells. The abundance of exonic transcript reads was determined by analysis of total RNA-seq data generated for pro-B and small pre-B cells from mice of the indicated genotypes before (–) and after (+) 5-Ph-IAA treatment. The regulation of Vpreb1 and Vpreb2 by Ikaros (8 h), Aiolos (8 h), and Ikaros plus Aiolos (8 h and 12 h) in pre-B cells is shown in the lower panel. The indicated DESeq2-derived fold changes and mean TPM values with SEM are based on two total RNA-seq experiments. The distantly related Vpreb3 protein37 may play only a minor role, if any, in pre-BCR formation, as Vpreb3 cannot compensate for the pre-BCR loss in Vpre1,Vpreb2 double-mutant mice83. d–f, Chromatin accessibility changes at putative enhancers of Irf4 (d), Bcl2l1 (e), and Ikzf1 (f) upon degradation of the indicated TFs in small pre-B cells. Binding of E2A, Ebf1, Pax5, and Ikaros was determined by CUT&RUN in the respective untreated pre-B cells, while open chromatin was determined by ATAC-seq analysis before (–) and after (+) 5-Ph-IAA treatment. A putative E2A- and Pax5-dependent enhancer is located 28 kb upstream of the Irf4 gene.

Extended Data Fig. 10 Regulation of the Igh locus in pro-B cells and summary of interactions of E2A, Ebf1, Pax5, Ikaros, and Aiolos with relevant target genes.

a, Chromatin accessibility, nascent transcript abundance, and binding of Ebf1 (upper part) and Pax5 (lower part) at the Igh locus. TF binding was determined by CUT&RUN analysis in untreated pro-B cells, while nascent transcripts and open chromatin were determined by total RNA-seq and ATAC-seq, respectively, before (–) and after (+) 5-Ph-IAA treatment. Known regulatory elements are indicated. b, Schematic diagram depicting extended loop extrusion across the entire 2.8-Mb long Igh locus upon Pax5-mediated repression of the cohesin-release factor Wapl in pro-B cells43. The cohesin complex (orange) generates chromatin loops by extruding DNA sequences at the Igh locus. The Rag1,2-bound recombination center (gray) is located at the DJH-rearranged gene segment in the 3′ proximal ___domain. Ongoing loop extrusion leads to convergent alignment of the 12-RSS (recognition signal sequence with a 12-bp spacer, black arrowhead) of the DH segment and the 23-RSS (green arrowhead) of the VH genes, which mediates RAG-cleavage and subsequent VH-DJH joining. c, Summary of the interactions of E2A, Ebf1, Pax5, Ikaros, and Aiolos with relevant target genes described in this publication. Gene activation and repression is shown in blue and red, respectively. Interactions in pro-B, pre-B, and immature B cells are indicated by dotted, dashed and full lines, respectively. Interactions are shown for regulated target genes (>1.5-fold). The transcriptional data of the following target genes are shown in the indicated figures: Cd19 (Extended Data Fig. 5b), Bcl2l1, Ikzf1 (Fig. 3b, c), Bach2, Cbfa2t3, E2f2, Ebf1, Myb, Myc, Pax5, Pou2af1, Tcf3 (Fig. 6), Ikzf3 (Extended Data Fig. 4b), Dntt, Igll1, Irf4, Irf8, Rag1, Rag2 (Fig. 7), Vpreb1 (Extended Data Fig. 9c), and Wapl (Fig. 8a).

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Supplementary Table 1

Direct target genes of E2A, Ebf1, Pax5, Ikaros and Aiolos in pro-B, small pre-B and immature B cells.

Supplementary Table 2

Comparison of Ikaros-regulated genes identified by Ikaros degradation and conditional Ikzf1 mutagenesis in pro-B cells.

Supplementary Table 3

Motif enrichment analysis based on the CUT&RUN data of the present study.

Supplementary Table 4

Regulation of transcriptional regulators by E2A, Ebf1, Pax5, Ikaros and Aiolos in pro-B, small pre-B and immature B cells.

Supplementary Table 5

Oligonucleotide sequences: genotyping primers, sgRNA for CRISPR–Cas9-mediated genome editing and MEDS oligonucleotides for ATAC-seq.

Supplementary Table 6

Description and GEO accession nos. of all Illumina sequencing experiments generated for the present study.

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Fedl, A.S., Tagoh, H., Gruenbacher, S. et al. Transcriptional function of E2A, Ebf1, Pax5, Ikaros and Aiolos analyzed by in vivo acute protein degradation in early B cell development. Nat Immunol 25, 1663–1677 (2024). https://doi.org/10.1038/s41590-024-01933-7

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