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. 2019 Jan 18;8:e43821. doi: 10.7554/eLife.43821

Figure 3. HDAC3-deficient thymocytes fail to induce the CD4-lineage program.

(A) Flow cytometric analysis of populations from OT-II and OT-II HDAC3-cKO mice sorted for RNA-seq and ChIP-seq. Plots show representative pre-sort and post-sort analysis of two FACS sorted populations—Immature thymocytes (Vβ5loCD69- CD4+CD8+) and Selecting thymocytes (Vβ5loCD69+). (B) Expression of CD4-lineage and CD8-lineage gene sets across DP, CD4+CD8lo, CD4SP, and CD8SP ImmGen expression data. (C–D) Volcano plot depicting the Log2fold change (FC) between OT-II HDAC3-cKO and OT-II Selecting (CD69+) cells. Grey dots show all genes; pink dots show CD4-lineage gene set (C); blue dots show CD8-lineage gene set (D). (E–F) Average H3K27ac ChIP-seq signal at the transcription start site (TSS) between OT-II and OT-II HDAC3-cKO Selecting (CD69+) cells for CD4-lineage gene sets (E) and CD8-lineage gene sets (F). Box-and-whisker plots depict H3K27ac signal at the TSS at CD4- or CD8-lineage genes between OT-II and OT-II HDAC3-cKO mice. Snapshots of example ChIP-seq tracks for each gene set are below. See also Figure 3—figure supplements 1–5 and Figure 3—source datas 1–2.

Figure 3—source data 1. Gene expression values (RNA-seq) of Immature and Selecting populations from OT-II and OT-II HDAC3-cKO mice.
Numerical data corresponding to the graphs of Figure 3C–D.
elife-43821-fig3-data1.xlsx (4.8MB, xlsx)
DOI: 10.7554/eLife.43821.010
Figure 3—source data 2. Gene lists: CD4-lineage, CD8-lineage, silenced genes, housekeeping genes.
CD4-lineage and CD8-lineage gene lists corresponding to graphs of Figure 3B–F. Silenced genes and housekeeping gene lists corresponding to graphs of Figure 3—figure supplement 5.
elife-43821-fig3-data2.xls (178.5KB, xls)
DOI: 10.7554/eLife.43821.011

Figure 3.

Figure 3—figure supplement 1. Characterization of Immature and Selecting thymic populations.

Figure 3—figure supplement 1.

(A) CD4-versus-CD8 profile of Immature (purple) and Selecting (green) populations in OT-II mice. Immature thymocytes are gated as Vβ5-/loCD69- CD4+CD8+. Selecting thymocytes are gated as Vβ5intCD69+. (B) Overlay of Selecting (blue) and CD4+CD8lo (red) populations on a Vβ5-versus-CD69 profile in OT-II mice. Grey dots on the Vβ5/CD69 plot depict total thymocytes. Data is representative of 3 mice from three independent experiments. (C) Selecting cells show intermediary marker expression between DP and CD4+CD8lo thymocytes. Expression of ‘down-regulated’ genes (CD8α, RORγt, Bcl-xl) and ‘up-regulated’ genes (CD69, TCR, CCR7, IL-7Rα) on DP, CD4+CD8lo, and Selecting cells from OT-II mice. Data is representative of 3 mice from three independent experiments.
Figure 3—figure supplement 2. Gene expression of select housekeeping genes, CD4-lineage genes, and CD8-lineage genes.

Figure 3—figure supplement 2.

RPKM values of indicated genes of Selecting cells from OT-II and OT-II HDAC3-cKO mice. (A) Housekeeping genes, (B) CD4-lineage genes, and (C) CD8-lineage genes. Bar graphs depict mean ± SEM of three mice. P values (***, p < 0.001, **, p < 0.01) were calculated using the exactTest with edgeR software. Housekeeping genes did not show a significant difference between mice. (D) Protein expression of ThPOK and Runx3 in Selecting cells from OT-II and OT-II HDAC3-cKO. Bar graphs depict mean ± SEM of 3–6 mice per group from at least three independent experiments. P values were calculated using paired t test.
Figure 3—figure supplement 3. Heatmaps of CD4-lineage and CD8-lineage genes.

Figure 3—figure supplement 3.

Heatmaps depict Log10 RPKM of genes in CD4-lineage (A) and CD8-lineage (B) gene sets in Selecting (CD69+) thymocytes from OT-II and OT-II HDAC3-cKO. Heatmaps show three biological replicates per mouse group. Scale bar is below each heatmap. Refer to Figure 3—source data 1 for Log2fold change between OT-II and OT-II HDAC3-cKO mice.
Figure 3—figure supplement 4. H3K9ac signal at CD4-lineage and CD8-lineage genes.

Figure 3—figure supplement 4.

(A–B) Average H3K9ac ChIP-seq signal at the transcription start site (TSS) between OT-II and OT-II HDAC3-cKO Selecting cells for CD4-lineage gene sets (A) and CD8-lineage gene sets (B). Box-and-whisker plots depict H3K9ac signal at the TSS at each CD4- or CD8-lineage gene between OT-II and OT-II HDAC3-cKO mice. Snapshots of example ChIP-seq tracks for each gene set are below plots depicting the average signal. P values were calculated using Wilcoxon signed rank test.
Figure 3—figure supplement 5. H3K9ac signal and H3K27ac signal at silenced genes and housekeeping genes in thymocytes.

Figure 3—figure supplement 5.

(A–B) Average H3K9ac and H3K27ac signal ±20 kb around the gene body among gene sets that are silenced in thymocytes (A) or housekeeping genes (B). Below are example H3K9ac ChIP-seq snapshots of genes within each of the genesets—Csf1r for macrophage, Pax5 for B cell, Cd34 for HSC/progenitor, Hprt for housekeeping, Atp5g1 for mitochondrial, and Rpl15 for ribosomal. Each image depicts an overlay of ChIP-seq tracks between OT-II (blue) and OT-II HDAC3-cKO (orange) mice. Refer to Figure 3—source data 2 for list of genes in these gene sets (macrophage, B cell, HSC/Progenitor, mitochondrial, ribosomal). TSS represents transcription start site; TES represents transcription end site. Scale bar in kb below ChIP-seq tracks identifies scale of snapshot.