Fig. 3: Nr4a1 and Nr4a3 are required for upregulation of Bcl2l11/BIM in SP thymocytes by self-Ag recognition. | Nature Communications

Fig. 3: Nr4a1 and Nr4a3 are required for upregulation of Bcl2l11/BIM in SP thymocytes by self-Ag recognition.

From: Nr4a1 and Nr4a3 redundantly control clonal deletion and contribute to an anergy-like transcriptome in auto-reactive thymocytes to impose tolerance in mice

Fig. 3

a Nr4a1 and Bcl2l11 transcript UMAP data from publicly available VISION thymus CITEseq interface. Red arrows indicate two clusters of negatively selecting cells, early in cortical DP and late in medullary SP. See also Supp Fig. 1e for additional annotation. b, c Nur77-eGFP BAC Tg thymocytes were stained to detect thymic subsets and intra-cellular BIM protein. Representative plots depict correlation between markers and show GFPlo/hi gates. Graph (c) depicts MFI of BIM among GFPhi and GFPlo populations as gated in (b) +/– SEM from 4 biological replicates. d, e Nur77-eGFP BAC Tg was crossed onto the Bcl2l11–/– background. Thymocytes from Bcl2l11 + /+ and –/– reporter mice were stained to detect SP subsets (d, top panels) and gating for pre-selection (CD69lo TCRβlo) and post-selection (CD69hiTCRβhi) DP thymocytes (d, bottom panels). Histograms depict reporter GFP expression in overlaid genotypes gated as in (d) along with peripheral CD4, CD8 LN T cells. Data are representative of 3 biological replicates. f, g Graphs depict RPKM for Gadd45b (f) and Bcl2l11 (g) in CD69hi Va2 + SP4 thymocytes of either WT-OT-II or DKO-OT-II donor genotypes from chimeras depicted in 3a. RNAseq was performed on biological replicates (Supplementary Data 1a depicts p value and fdr for all pairwise comparisons across sample types, GSE235101). h, i Overlaid histograms depict intracellular BIM protein expression in SP4 OT-II and DKO-OT-II thymocytes from N = 4 WT host and N = 3 RIPmOVA host chimeras – biological replicates (as schematized in 3a). Graph (i) depicts BIM MFI + /- SEM from these samples. j, k Overlaid histograms depict intracellular BIM protein expression in CD45.2 donor genotypes in SP4 (top) and SP8 (bottom) thymocytes from N = 3 control WT:WT chimeras and N = 4 experimental DKO:WT polyclonal chimeras – biological replicates (as schematized in 1c). Graphs (k) depict BIM MFI across thymic subsets in both CD45.2 and CD45.1 donor genotypes from both types of chimeras. Lines connect samples from the same chimera. l UCSC browser tracks depict ATACseq peaks from public immgen.org data across thymic subsets and Follicular splenic B cells for reference at the Acoxl/Bcl2l11 locus. 200 kb upstream of the TSS of Bcl2l11 are green-tinted ATAC peaks with consensus Nr4a sites that correspond to EBAB enhancer identified in Hojo et al. (PMID 31197149). Blue tint identifies an adjacent peak with striking cell type specificity but only an imperfect Nr4a site. See also Supplementary Data 1b. m Graph depicts RPKM for Acoxl as in f, g, statistical analyses in Supplementary Data-Table 1a. Statistical tests: c, k unpaired parametric two-tailed t-test, assume equal SD. i one way ANOVA with Tukey’s multiple hypothesis test. Source data are provided as a Source Data file.

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