During the germinal center (GC) reaction, B cells undergo profound transcriptional, epigenetic and genomic architectural changes. How such changes are established remains unknown. Mapping chromatin accessibility during the humoral immune response, we show that OCT2 was the dominant transcription factor linked to differential accessibility of GC regulatory elements. Silent chromatin regions destined to become GC-specific super-enhancers (SEs) contained pre-positioned OCT2-binding sites in naive B cells (NBs). These preloaded SE ‘seeds’ featured spatial clustering of regulatory elements enriched in OCT2 DNA-binding motifs that became heavily loaded with OCT2 and its GC-specific coactivator OCAB in GC B cells (GCBs). SEs with high abundance of pre-positioned OCT2 binding preferentially formed long-range chromatin contacts in GCs, to support expression of GC-specifying factors. Gain in accessibility and architectural interactivity of these regions were dependent on recruitment of OCAB. Pre-positioning key regulators at SEs may represent a broadly used strategy for facilitating rapid cell fate transitions.
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A.S.D. is funded by NCI (1 F31 CA220981-01). A.M.M. and O.E. are funded by Leukemia and Lymphoma Society SCOR 7012-16 and SCOR 7021-20. O.E. is supported by Janssen and Eli Lilly research grants, NIH grants UL1TR002384, R01CA194547 and LLS SCOR grant 180078-02. A.M.M. is funded by NCI R35 CA220499, and NCI P01 CA229086, LLS TRP 6572-19, The Chemotherapy Foundation and the Follicular Lymphoma Consortium. R.G.R. is supported by NIH grants R01AI148387 and R01CA178765 and Leukemia and Lymphoma Society SCOR 7021-20. This study makes use of data generated by the Blueprint Consortium. A full list of the investigators who contributed to the generation of the data is available from www.blueprint-epigenome.eu. Funding for the project was provided by the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no 282510 BLUEPRINT.
A.M.M. receives research funding from Janssen and Daiichi Sankyo, has consulted for Constellation, Jubilant and Epizyme, and is on the advisory board of KDAC. O.E. is scientific advisor and equity holder in Freenome, Owkin, Volastra Therapeutics and OneThree Biotech. A.S.D., C.-S.C., D.C.D.G., M.A.R., J.C.H., Y.J., N.Y., A.A., R.G.R. and E.A. declare no competing interests.
Peer review information Nature Immunology thanks Marcus Clark, Rhys Allan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Figure 1a, gating strategy used for the isolation of naïve B cells (NB), memory B cells (MB), plasmablasts (PC), germinal center centroblasts (CB), and germinal center centrocytes (CC) from human tonsillar lymphocytes using multiparameter fluorescence activated cell sorting (FACS). Lymphocytes were isolated from fresh human tonsils by density gradient centrifugation. Within 24 hours, cells were either sorted by FACS or cryopreserved for cell sorting on a later date (see Methods). Prior to sorting, lymphocytes were labeled with anti-CD20, anti-CD10, anti-CD44, anti-CD27, anti-CD38, and anti-CXCR4 conjugated flourochromes. DAPI was used to exclude non-viable cells. For isolation of NBs, centroblasts, and centrocytes only, anti-IgD was used in place of anti-CD38 and anti-CD27, and NBs were CD20+CD44+CD10−IgD+. Details of antibodies used for cell sorting are listed in Extended data Table 1. SSC: side scatter, FSC: forward scatter.
Extended Data Figure 2a, ATAC-seq insertion profiles in GCBs for 2kb regions centered on gene transcription start sites. Mean profile is shown for genes by quintile of mRNA abundance in GCBs. mRNA abundance represented as TPM (transcripts per kilobase million) as estimated by Salmon58. b, NucleoATAC42 estimates of nucleosome position and occupancy for gene TSS according to a gene’s mRNA abundance quintile in GCBs. c, MNAse-seq reads per million mapped reads (RPM) according to a gene’s mRNA abundance quintile in GCBs. d, NucleoATAC estimates for the position of +1 nucleosome relative to the TSS of genes according to a gene’s mRNA abundance quintile in GCB (n = 10 biological replicates). e, Density plots showing dynamic range of normalized ATAC-seq read counts in ATAC-seq peak-atlas sites across B-cell populations. Promoter elements are located < 2kb from a gene TSS. Bars under density plots indicate inter-quantile range. f, principal components analysis (PCA) of ATAC-seq insertion counts in gene-distal peak-atlas elements across B cell populations. g, Cumulative probability distribution plot of GCB (n = 13) vs NB (n = 9) gene expression log2-fold changes (RNA-seq) for genes assigned to DNA elements that are differentially accessible (FDR < 0.01) or unchanged in GCB (n = 10) vs NB (n = 6). Gene expression log2 fold changes are computed using DESEq261. P-values are calculated by Wilcoxon rank-sum test and bars adjacent to p-values indicate the comparison of newly accessible or decommissioned sites to unchanged sites.
Extended Data Figure 3a, results of de novo motif discovery using GCB OCT2 peak summits as input to HOMER62. b, bar plot showing the fraction of GCB de novo accessible or GCB decommissioned sites with or without OCT2 binding. P-values are calculated by Fisher’s exact test. c, scatter plot showing Bayesian posterior predicted mean chromatin accessibility log2 fold-changes in GCB (n = 10) vs NB (n = 6) for sites with 0, 1, 2, or > = 3 OCT2 motifs, and located >5 kb or < 5kb of a gene TSS. P-value is computed by ANOVA F-test from observed data, and error bars indicate 99% credible intervals. d, bar plot showing sites by their distance to a gene TSS for differentially accessible (FDR < 0.001) or unchanged sites in mouse GCBs (n = 10) vs NBs (n = 4). P-values are calculated by Fisher’s exact test. e, scatter plot showing the mean accessibility log2 fold change in mouse GCBs vs NBs for promoter and distal DNA elements with or without an OCT2 motif. P-value is calculated by ANOVA testing the null hypothesis that the difference in accessibility log2 fold-changes between promoter and distal elements does not depend on the presence of an OCT2 DNA motif. Error bars indicate 95% CIs. f, TF-motif effect size estimates for change in accessibility in mouse GCBs (n = 4) vs NBs (n = 4). Motif logos represent motif position weight matrices used. g, TF-motif effect sizes for histone H3K4me1 log2 fold-changes in mouse GCBs (n = 4) relative to NBs (n = 4); h, for histone H3K27ac log2 fold-changes in mouse GCBs (n = 4) vs NBs (n = 4); and i, for gene expression log2 fold-changes in mouse GCBs (n = 7) vs NBs (n = 6). Error bars represent 95% CIs. j, immuno blot for OCAB and OCT2 in human NBs and GCBs. This blot was repeated independently with similar results for two biological replicate samples.
Extended Data Figure 4a, results of de novo motif discovery as reported by HOMER for OCT2 ChIP-seq NBs. b, bar plots comparing the genomic localization of indirect, pre-positioned, and GCB de novo OCT2 sites. c, genome browser plot of OCT2 ChIP-seq in NBs and GCBs, OCAB ChIP-seq in GCBs, and ATAC-seq in NBs and CBs. ChIP-seq tracks show fold enrichment/input and each sample is plotted using identical Y scale. ATAC-seq tracks were normalized to number of reads B cell DNA elements and use identical Y scales across samples. d, density plots showing the mean distance to the nearest GCB de novo OCT2 site for pre-positioned OCT2 sites (red) or random sample of DNA elements in B cells (expectation, gray). Statistics were computed by resampling using the regioneR package in R. e, Box plot comparing cluster coreness for differentially accessible or unchanged sites in GCBs (n = 10) vs NBs (n = 6). Cluster coreness of sites is computed using a graph of peak-peak distances with clusters defined by infonet community detection. P-values are computed by Mann-Whitney U test. f, ATAC-seq insertion profiles in GCB and NB centered on the OCT2 motif in pre-positioned and GCB de novo OCT2 sites. g, boxplot comparing H3k27ac activating mark and H3K27me3 repressive mark in NB and GCB at OCT2 pre-positioned sites. P-values are calculated by Mann-Whitney U test. h, read density heat maps showing ChIP-seq enrichment/input for OCT2, OCAB, and histones H3K4me1 and H3K27ac. ATAC-seq heatmaps show log2 (GCB / NB) insertions. Box plots show center line as median, box limits as upper and lower quartiles, whiskers as minimum and maximum values within 1.5 x inter-quartile range of 1st and 3rd quartile, and notches as approximate 95% CI of the median.
Extended Data Figure 5a, comparison of motif position weight matrices as defined from de novo motif discovery of pre-positioned and GCB de novo sites. Motif divergence was computed using the R package motifDiverge. Significant divergence indicated by *. b, results of de novo motif discovery as reported by HOMER for OCT2 ChIP-seq and OCAB ChIP-seq in GCBs. c, barplot showing the fraction of sites with OCAB recruitment in mouse GCBs and comparing sites with OCT2 direct binding in murine splenic B cells and sites in which OCT2 is absent. ChIP-seq data for OCT2 and OCAB were retrieved from GSE142040. P-value by Fisher’s exact test. d, boxplots of log2 fold changes in mouse GCB (n = 10) vs NB (n = 4) chromatin accessibility, mouse GCB (n = 2) vs NB (n = 2) histone H3K27ac, and mouse GCB (n = 2) vs NB (n = 2) histone H3K4me1 and comparing OCT2 direct sites in splenic B cells that recruit OCAB in GCBs (OCT2-OCAB sites) with all other accessible sites. P-values by Welch t-test. Box plots show center line as median, box limits as upper and lower quartiles, whiskers as minimum and maximum values within 1.5 x inter-quartile range of 1st and 3rd quartile, and notches as approximate 95% CI of the median.
Extended Data Figure 6a, illustration of the strategy for OCAB depletion (OCABi) in GCB-derived cells. Viable, GFP+ OCI-Ly7 and SUDHL4 cells expressing dCas9-KRAB were isolated by FACS 72 hours after transduction with sgRNA and GFP. b, chromatin accessibility PCA of B cells and cell lines using DAEs across B cell phenotypes. c, gene expression PCA of B cells and cell lines using differentially expressed genes across B cell phenotypes (FDR < 0.001). d, plot showing the fraction of GFP+ viable OCI-Ly7 or SUDHL4 (e) cells after transduction with control sgRNAs (n = 6) or sgRNAs targeting OCAB (OCABi, n = 15). Shaded area shows 95% CI of the mean. f, bar plot showing OCAB transcript abundance (TPM)51 in OCABi (n = 6 replicate experiments) vs control (n = 6) OCI-Ly7 cells, and OCABi (n = 2) vs control (n = 2) SUDHL4 cells. P-values are calculated by Welch t-test and error bars show standard error of the mean. g, OCAB expression relative to HPRT in control (n = 3) and OCABi (n = 3) OCI-Ly7 cells by qPCR. P-values are calculated by Welch t-test and error bars show standard error of the mean. h, unsupervised hierarchical clustering dendrogram based on Pearson correlation distance of OCABi and control OCI-Ly7 and SUDHL4 chromatin accessibility and gene expression (i). j, scatter plot showing chromatin accessibility of OCABi and control OCI-Ly7 or SUDHL4 (k) cells projected onto PCs from primary B cell chromatin accessibility PCA (Fig. 4I). l, scatter plot showing OCI-Ly7 OCABi and control or SUDHL4 OCABi and control (m) gene expression projected onto PCs from primary B cells (Fig. 4K). n, illustration showing trajectories in B-cell development during the humoral immune response. Relative position of cell types and length of arcs is defined from PCA of mean accessibility signal for each cell type.
Extended Data Figure 7a, experimental design for forced expression of Pou2af1, the gene encoding OCAB in mouse. Splenic B cells from n = 5 mice were stimulated with LPS ex vivo and transduced with a GFP vector containing Pou2af1 (mOCAB) or GFP only (control). b, expression of Pou2af1 in mOCAB (n = 5) and control(n = 5) B cells. Expression levels were measured by qPCR using cDNA generated from total RNA of GFP+ viable cells and normalized to that of Hprt1, calculated by the delta-delta Ct method (2−ΔΔCtand represented as a fold change over the mean expression in control cells. The expression of each gene was measured in triplicate across 5 replicates. P-values are calculated by Welch’s t test and error bars show standard error of the mean.
Extended Data Figure 8a, pie chart showing the fraction of H3K27ac HiChIP loops that form enhancer-promoter (E-P) contacts for loops anchored by an OCAB-OCT2 sites (left) or not anchored by an OCAB-OCT2 site (right). The inner circle represents the fraction of loops that form E-P contacts, and the outer circle distinguishes whether the promoter belongs to a gene whose expression is differentially increased, decreased, or unchanged in GCB vs NB. *Indicates significantly greater fraction for OCT2-OCAB loops compared to loops not anchored by OCT2-OCAB. P-values by Fisher’s exact test and * indicates FDR-corrected p < 10−5. b, effect estimates for the contribution of enhancer activity score to a gene’s change in expression in biological replicate GCBs (n = 6) relative to NBs (n = 6) for enhancers with direct binding present or direct binding absent for the indicated TF. Effect sizes were computed from a linear regression of GCB / NB expression log2 fold-change on the total enhancer activity score per gene for enhancers with and without TF direct binding, and error bars show 95% CI for mean effect estimate. Activity-contact score was computed from OCI-Ly7 H3K27ac HiChIP (n = 2 biological replicates), GCB H3K27ac ChIP-seq (n = 4 biological replicates), and using the 76237 ATAC-seq DNA elements identified in B cells. c, APA plots showing aggregate contact enrichment vs local background in Control and OCABi OCI-Ly7 cells and the difference in contact enrichment between OCABi and Control. Plots centered on anchor mid-points of called loops that were sensitive to OCAB depletion at 1% FDR.
Extended Data Figure 9a, Box plots of cluster coreness comparing DNA elements with OCT2-OCAB (n = 6768) or without OCT2-OCAB (n = 69469). P-values are calculated by Mann-Whitney U test. Box plots show center line as median, box limits as upper and lower quartiles, and whiskers as minimum and maximum values within 1.5 x inter-quartile range of 1st and 3rd quartile. b, box plots showing log2 fold change in histone H3K27ac ChIP-seq enrichment in GCBs (n = 4) vs NBs (n = 4) for clustered sites with or without OCT2-OCAB binding. P-value is computed by Welch’s t-test. c, genome browser plot of the BCL6 locus. d, Venn diagram of super-enhancers (SEs) in NBs and GCBs. e, plot of enhancers and SEs (red points) in GCBs. f, empirical cumulative distribution plot of contact distance for EP loops. P-value is calculated by Mann-Whitney U test. g, APA plots of GC-activated SE loops in control and OCABi OCI-Ly7 cells. h, bar plot of H3K27ac HiChIP APA scores for 621 GC-activated SE loops in OCI-Ly7 control (n = 3) or OCABi (n = 2) cells. P-value is calculated by t-test and error bars show standard error of the mean. i, GSEA enrichment plot using expression change in OCABi vs control for target genes of GC-activated SEs. j, Scatter plot showing per-gene aggregate HiChIP contact enrichment log2 fold changes in OCI-Ly7 OCABi vs Control. k, Scatter plot of aggregate H3K27ac ChIP-seq log2 fold changes in GCBs vs NBs for sites within a SE. Red and blue points indicate SEs above the 90th and below the 10th percentile, respectively. l, Super-enhancer analysis using the ROSE method with GCB OCT2 ChIP-seq signal over stitched GCB OCT2 ChIP-seq peaks. m, scatter plot showing the number of called H3K27ac Hi-ChIP enhancer-promoter loops per gene promoter for genes expressed in OCI-Ly7 cells.
Extended Data Figure 10a, genome browser plot of a region on chromosome 3 containing BCL6 and adjacent super-enhancer. OCT2-OCAB sites are shown with Pre-positioned OCT2 sites in red and GCB de novo OCT2 sites in black. H3K27ac HiChIP called loops in OCI-Ly7 cells. Plot showing NB and GCB 4C contact enrichment representing interactions with the 4C anchor, located at the BCL6 TSS. Bar plot showing differential 4C contact enrichment with bars labeled according to FDR for differential contacts. Normalized and differential contact enrichment computed using 4C-ker43 and DESeq261.
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Doane, A.S., Chu, CS., Di Giammartino, D.C. et al. OCT2 pre-positioning facilitates cell fate transition and chromatin architecture changes in humoral immunity. Nat Immunol 22, 1327–1340 (2021). https://doi.org/10.1038/s41590-021-01025-w