Journal: Nature Communications

Article Title: Setd2 supports GATA3 + ST2 + thymic-derived Treg cells and suppresses intestinal inflammation

doi: 10.1038/s41467-022-35250-0

Figure Lengend Snippet: a , b H3K36me3 ChIP-seq analysis on Treg cells (CD3 + CD4 + Foxp3-YFP + ) sorted from spleen (SP) or large intestine (LI) of Foxp3 Cre-YFP mice. a Cumulative curve of average H3K36me3 signals distributed scaled regions of all genes, SPTreg signature genes and Colonic Treg signature genes. TSS transcription start site, TES transcription end site. b Correlation analysis on all SPTreg and Colonic Treg signature genes (GSE68009) using Log 2 (fold change) (Fc) of gene mRNA expression and Log 2 (Fc) of summed H3K36me3 signals. Each dot represents one gene. Percentages indicate the proportions of genes distributed in each quadrant. R is Pearson correlation coefficient. c , d LI Treg cells sorted from Foxp3 Cre-YFP Setd2 f/+ (Ctrl) or Foxp3 Cre-YFP Setd2 f/f (KO) mice were subjected to RNA-seq analysis. c Heatmap showing differentially expressed genes based on Z -score of normalized FPKM value, and corresponding normalized Log 2 Fc of LITreg over SPTreg in microarray data of GSE68009 and Log 2 Fc of summed H3K36me3 signals in LITreg over SPTreg. d GSEA analysis based on ST2 + Treg cells signature. e – k Cells were isolated from the LI. e , f Flow cytometry analysis of expression of RORγt and Helios gated on Treg cells (CD3 + CD4 + Foxp3 + ). f Percentage of Helios – RORγt + cells. (Ctrl n = 8; KO n = 7). g , h Flow cytometry analysis of ST2 and Helios expression gated on Treg cells. ( n = 7 per group). h Percentages of DP Treg cells (double positive, Helios + ST2 + cells) and ST2 Single Treg cells (ST2 single positive, Helios – ST2 + cells). i , j , k Flow cytometry analysis of GATA3 and Helios expression gated on Treg cells. (Ctrl n = 6; KO n = 8. For analyzing total cell number, n = 6 per group). Percentages ( j ) and absolute numbers ( k ) of DP Treg cells (Helios + GATA3 + ), Helios Single Treg cells (Helios + GATA3 – ), GATA3 Single Treg cells (Helios – GATA3 + ), and DN Treg cells (double negative, Helios – GATA3 – ). l , m Percentages and numbers of Helios + ST2 + Treg cells ( l ) and Helios + GATA3 + Treg cells ( m ) in indicated organs analyzed by flow cytometry. ( n = 4 per group). f , h , j – m Representative of 2–4 independent experiments. Data were means ± SEM. Source data are provided as a Source Data file.

Article Snippet: Pearson correlation analysis was performed with GraphPad Prism 8.0.

Techniques: ChIP-sequencing, Expressing, RNA Sequencing, Microarray, Isolation, Flow Cytometry

Journal: Nature Communications

Article Title: Setd2 supports GATA3 + ST2 + thymic-derived Treg cells and suppresses intestinal inflammation

doi: 10.1038/s41467-022-35250-0

Figure Lengend Snippet: a – c H3K27ac CUT&Tag analysis was performed with Treg cells purified from 2-week-old large intestinal LPLs of Foxp3 Cre-YFP Setd2 f/+ (Ctrl) or Foxp3 Cre-YFP Setd2 f/f (KO) mice. d , e Genome browser tracks of H3K27ac, RNA Pol II CUT&Tag peaks, H3K36me3 peaks with published Treg H3K4me1(DRP003376) peaks at the Il1rl1 and Gata3 locus. Boxes highlight differentially expressed peaks with or close to reach statistically significant differences analyzed by DEseq2. f , g RNA Pol II CUT&Tag analysis was performed with splenic Treg cells nuclei of IL-2c-treated Foxp3 Cre-YFP Setd2 f/+ (Ctrl) or Foxp3 Cre-YFP Setd2 f/f (KO) mice. a – c , f , g Correlation analysis was performed on top significantly changed genes identified by intestinal Treg RNA-seq using Log 2 (fold change) (Fc) of gene mRNA expression and average Log 2 (fold change) (Fc) of H3K27ac peaks distributed at the promoters ( a ), H3K27ac peaks distributed at gene bodies ( b ), H3K27ac peaks distributed at intergenic regions ( c ), Pol II peaks distributed at the promoters ( f ), and Pol II peaks distributed at gene bodies ( g ). a – c , f , g Each dot represents one gene. Percentages indicate the proportions of genes distributed in each quadrant. R is the Pearson correlation coefficient. NS not significant. h , i Treg cells (CD3 + CD4 + YFP + ) were sorted from the spleen of IL-2c-treated mice of indicated genotypes. mRNA expression of Il1rl1 ( h ) and Gata3 ( i ) were analyzed by real-time RT-PCR ( n = 3 per group). h , i Representative of two independent experiments. Data were means ± SEM. Source data are provided as a Source Data file.

Article Snippet: Pearson correlation analysis was performed with GraphPad Prism 8.0.

Techniques: Purification, RNA Sequencing, Expressing, Quantitative RT-PCR