Journal: eLife

Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4 + CD8 + thymocytes for CD4-lineage commitment

doi: 10.7554/eLife.43821

Figure Lengend Snippet: ( A ) CD4/CD8 profile of mature thymic SP (Vβ5 + Vα2 + H2K + CD24 lo ) and splenic T cells (Vβ5 + Vα2 + ) from OT-II, OT-II RORγt-KO Bcl-xl tg (RB) and OT-II RB3 mice. Graphs depict frequency of gated cells from at least three independent experiments (n = 3–5/group) ( B ) Runx3 and ThPOK expression in mature thymic SP cells from OT-II, OT-II RB, and OT-II RB3 mice (n = 4/group from four independent experiments). ( C ) CD4/CD8 profile of mature thymic SP from OT-II RORγt-KO and OT-II RORγt-KO HDAC3-cKO mice. Graphs depict frequency of gated cells from at least three independent experiments (n = 3–4/group). ( D ) Granzyme b and perforin expression in SP thymocytes from OT-II, OT-I, OT-II RB and OT-II RB3 mice. FACS plots were gated on CD4SP cells for OT-II and OT-II RB mice and CD8SP cells for OT-I and OT-II RB3 mice. Bar graphs depict mean ± SEM (n = 3/group from three independent experiments) of the fold change in MFI between unstimulated and TCR/CD2 stimulated conditions. ( E ) Representative FACS plots of the proportion of CD4SP and CD8SP mature thymocytes from straight BMCs, where bone marrow from OT-II or OT-II RB3 mice were transplanted into B6.SJL or β2m-KO recipients. Mature SP thymocytes were gated as Vβ5 + Vα2 + H2K + CD24 lo . Mice (n = 3–5/group from three independent experiments) were analyzed 8–10 weeks after transfer. ( F ) Representative FACS plots of the proportion of CD4SP and CD8SP mature thymocytes from mixed BMCs from OT-II (CD45.1 - )/B6.SJL (CD45.1 + ) and OT-II RB3 (CD45.1 - )/B6.SJL (CD45.1 + ) mice. Mature SP thymocytes from CD45.1 + cells were gated as H2K + CD24 lo ; mature SP thymocytes from CD45.1 - cells were gated as Vβ5 + Vα2 + H2K + CD24 lo . Mice were analyzed 8–10 weeks after transfer. (n = 5/group from three independent experiments).

Article Snippet: Graphs depict fold enrichment over regions without HDAC3 binding ( Rpl30 primers, Cell Signaling Technologies (#7015)).

Techniques: Expressing

Journal: eLife

Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4 + CD8 + thymocytes for CD4-lineage commitment

doi: 10.7554/eLife.43821

Figure Lengend Snippet: ( 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 (Vβ5 lo CD69 - CD4 + CD8 + ) and Selecting thymocytes (Vβ5 lo CD69 + ). ( B ) Expression of CD4-lineage and CD8-lineage gene sets across DP, CD4 + CD8 lo , CD4SP, and CD8SP ImmGen expression data. ( C–D ) Volcano plot depicting the Log fold 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 – and – . 10.7554/eLife.43821.010 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 . 10.7554/eLife.43821.011 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 . Silenced genes and housekeeping gene lists corresponding to graphs of .

Article Snippet: Graphs depict fold enrichment over regions without HDAC3 binding ( Rpl30 primers, Cell Signaling Technologies (#7015)).

Techniques: RNA Sequencing Assay, ChIP-sequencing, Expressing, Whisker Assay

Journal: eLife

Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4 + CD8 + thymocytes for CD4-lineage commitment

doi: 10.7554/eLife.43821

Figure Lengend Snippet: 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.

Article Snippet: Graphs depict fold enrichment over regions without HDAC3 binding ( Rpl30 primers, Cell Signaling Technologies (#7015)).

Techniques: Software, Expressing

Journal: eLife

Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4 + CD8 + thymocytes for CD4-lineage commitment

doi: 10.7554/eLife.43821

Figure Lengend Snippet: Heatmaps depict Log 10 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 for Log 2 fold change between OT-II and OT-II HDAC3-cKO mice.

Article Snippet: Graphs depict fold enrichment over regions without HDAC3 binding ( Rpl30 primers, Cell Signaling Technologies (#7015)).

Techniques:

Journal: eLife

Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4 + CD8 + thymocytes for CD4-lineage commitment

doi: 10.7554/eLife.43821

Figure Lengend Snippet: ( 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.

Article Snippet: Graphs depict fold enrichment over regions without HDAC3 binding ( Rpl30 primers, Cell Signaling Technologies (#7015)).

Techniques: ChIP-sequencing, Whisker Assay

Journal: eLife

Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4 + CD8 + thymocytes for CD4-lineage commitment

doi: 10.7554/eLife.43821

Figure Lengend Snippet: ( 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 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.

Article Snippet: Graphs depict fold enrichment over regions without HDAC3 binding ( Rpl30 primers, Cell Signaling Technologies (#7015)).

Techniques: ChIP-sequencing

Journal: eLife

Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4 + CD8 + thymocytes for CD4-lineage commitment

doi: 10.7554/eLife.43821

Figure Lengend Snippet: ( A ) Surface expression of cytokine receptor chains γc, IL-4Rα, IL-7Rα, IL-15Rα, IL-2Rβ, and IL-21R on DP thymocytes from WT and HDAC3-cKO mice. Flow cytometric plots have an isotype control to illustrate background level of expression. ( B ) Gene expression (RNA-seq) of Il2rg , Il4ra, Il7r, Il15ra, Il2rb , and Il21r in Immature (DP) cells from OT-II and OT-II HDAC3-cKO mice. ( C ) Snapshot of H3K27ac ChIP-seq tracks at the Il4ra and Il21r gene loci in Immature (DP) thymocytes from OT-II and OT-II HDAC3-cKO mice. Shaded regions identify super-enhancers. ( D ) Protein expression of IL-21R on DP, CD4SP and CD8SP thymocytes from WT mice and DP thymocytes from HDAC3-cKO mice. ( E ) pSTAT5 levels in DP thymocytes from WT and HDAC3-cKO mice after 10 min ex vivo stimulation with IL-21, IL-15, IL-7, IL4, or media alone. ( F ) Protein expression of STAT5a and STAT5b in DP thymocytes from WT and HDAC3-cKO mice. ( G ) pSTAT3 and pSTAT1 levels after a 10 min in vitro IL-21 stimulation, and pSTAT6 levels after IL-4 stimulation of DP thymocytes from WT and HDAC3-cKO mice. ( H ) Quantitative ChIP (qChIP) of HDAC3 binding at the Il21r promoter in DP thymocytes from WT and HDAC3-cKO mice. Graph depicts fold enrichment over Rpl30 (n = 4 mice/group from four indpendent experiments). ( I ) DNase-seq and Hi-C arc plots at the Il4ra and Il21r gene loci in DP thymocytes and pooled DN3-to-DP thymocytes, respectively. Shaded region highlights where HDAC3 binds, as shown in . (DHS, DNA hypersensitivity sites). ( A, E, G ) Bar graph shows mean ± SEM of MFI from 4 to 5 mice from at least three independent experiments. ( D, H ) Plots are representative of at least three mice from three independent experiments. ( F ) Bar graph shows mean ± SEM of MFI from three mice from two independent experiments. (***, p < 0.001). See also – .

Article Snippet: Graphs depict fold enrichment over regions without HDAC3 binding ( Rpl30 primers, Cell Signaling Technologies (#7015)).

Techniques: Expressing, RNA Sequencing Assay, ChIP-sequencing, Ex Vivo, In Vitro, Binding Assay, Hi-C

Journal: eLife

Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4 + CD8 + thymocytes for CD4-lineage commitment

doi: 10.7554/eLife.43821

Figure Lengend Snippet: ( A ) IL-21R expression in DN (CD4 - CD8 - ), immature SP (ISP; CD4 - CD8 + TCRβ - ), and DP (CD4 + CD8 + ) thymocytes from WT and HDAC3-cKO mice. Bar graph is mean ±SEM of MFI. N = 3 mice/group. ( B ) p-STAT5 expression in ISPs from WT and CD2-icre HDAC3-cKO mice that were stimulated with the indicated cytokines for 10mins. Experimental conditions are the same as performed in . Bar graph shows mean ±SEM of p-STAT5 MFI fold to unstimulated conditions. N = 4–5 mice/group.

Article Snippet: Graphs depict fold enrichment over regions without HDAC3 binding ( Rpl30 primers, Cell Signaling Technologies (#7015)).

Techniques: Expressing

Journal: eLife

Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4 + CD8 + thymocytes for CD4-lineage commitment

doi: 10.7554/eLife.43821

Figure Lengend Snippet: ChIP-seq snapshots at Runx3 and Patz1 of publicly available HDAC3 ChIP-seq datasets in pro-B cells (GEO: GSM2096648, genome alignment mm10) and human CD4 T cells (GEO: GSM393952, genome alignment hg18). Alongside the HDAC3 ChIP-seq, DNase-seq (GEO: GSM2195840, genome alignment mm10) and H3K27ac ChIP seq from Immature OT-II and OT-II HDAC3-cKO thymocytes (as shown in ) were provided to visualize where HDAC3 binds in relation to regulatory elements and changes in histone acetylation between WT and HDAC3-cKO mice, respectively. Orange shaded regions demarcate super enhancers (SE) and blue shaded regions highlight where primers were made for qPCR. In pro-B cells, HDAC3 binds 50 kb upstream of Runx3 , at the Runx3 promoter, and approximately 2 kb downstream of the Patz1 promoter. Likewise, HDAC3 binds approximately 57 kb upstream of Runx3 and 2 kb downstream of the Patz1 promoter in human CD4 T cells. These three regions were used to make primers for HDAC3 qChIP performed in .

Article Snippet: Graphs depict fold enrichment over regions without HDAC3 binding ( Rpl30 primers, Cell Signaling Technologies (#7015)).

Techniques: ChIP-sequencing

Journal: eLife

Article Title: HDAC3 restrains CD8-lineage genes to maintain a bi-potential state in CD4 + CD8 + thymocytes for CD4-lineage commitment

doi: 10.7554/eLife.43821

Figure Lengend Snippet: ( A ) HDAC3 qChIP in DP thymocytes from WT and HDAC3-cKO mice. Plots show mean ± SEM of fold enrichment over Rpl30 (n = 3 mice/group from three independent experiments). ( B ) DNase-seq and Hi-C arc plots at the Runx3 and Patz1 gene loci in DP thymocytes and pooled DN3-to-DP thymocytes, respectively. Shaded region highlights where HDAC3 binds, as shown in ( A ). (DHS, DNA hypersensitivity sites). ( C ) HDAC3 protein expression measured by flow cytometry in DP, CD4 + CD8 lo , CD4SP, and CD8SP thymocytes from WT mice. Plots show mean ± SEM of fold enrichment over isotype (n = 4 mice/group from three independent experiments). ( D ) HDAC3 qChIP in SP thymocytes from OT-II and OT-I mice. Plots show mean ± SEM of fold enrichment over Rpl30 (n = 4 mice/group from two independent experiments). See also – .

Article Snippet: Graphs depict fold enrichment over regions without HDAC3 binding ( Rpl30 primers, Cell Signaling Technologies (#7015)).

Techniques: Hi-C, Expressing, Flow Cytometry

Journal: Journal of Lipid Research

Article Title: FTO mediates cell-autonomous effects on adipogenesis and adipocyte lipid content by regulating gene expression via 6mA DNA modifications

doi: 10.1194/jlr.M085555

Figure Lengend Snippet: Fto knockdown regulates 6mA DNA methylation at the Cebpd promoter. qPCR of 6mA-modified Rpl30, Gapdh, and L1Md-Gf-17 (A, C) and Cebpd promoter-containing DNA (B, D) in lysates of 2 day postconfluent 3T3-L1 preadipocytes in which Fto had been knocked down prior to confluence, after pulldown with anti-6mA antibody, normalized to transcript levels in the input sample (A, B) and calculated as fold-change (C, D). All plots represent the mean ± SEM for n = 4 per condition; unpaired t-test. **P < 0.01, ***P < 0.001.

Article Snippet: Rpl30 (intron 2) primers were from Cell Signaling Technologies.

Techniques: DNA Methylation Assay, Modification