Additional file 1 of Genetic and epigenetic regulation of Treg cell fitness by autism-related chromatin remodeler CHD8

Additional File 1: Fig. 1. Verification of deletion of Chd8 in Tregs. (A) Schema of mouse crossing to generate Chd8-/- mice. (B) Splenic Tregs (CD4+CD25+) were isolated from both genotypes of mice with a CD4+CD25+ regulatory T cell isolation kit. Cell purity was confirmed by FACS (B). The cells were used for PCR genotyping with primers for WT, Flox and KO allele of Chd8 or Foxp3-Cre (C). Total RNA was extracted from Tregs for qPCR to confirm deletion of Chd8. The mRNA levels of Chd8 are shown (arbitrary unit). The data are normalized to an 18S reference and expressed as mean plus SD of triplicates representative of two separated experiments (D). **P < 0.01 vs. WT control group. Fig. 2. CHD8 deficiency increases CD8+ T cells. Splenocytes were analyzed for CD4+ and CD8+ T cells by FACS. Representative dot plots are shown. The proportions and absolute numbers of CD4+ and CD8+ cells are summarized in bar graphs (mean plus SD) (WT: 6 males + 6 females; Chd8-/-: 4 males + 6 females). *P < 0.05, **P < 0.01 vs. WT control group. Fig. 3. CHD8 deficiency does not affect overt thymocyte and thymic Treg development. Thymocytes were analyzed for CD4-CD8- (DN), CD4+CD8+ (DP), CD4+CD8- (SP4), CD4-CD8+ (SP8) (A), and CD4+Foxp3+ (B) cells by FACS. Representative dot plots are shown. The proportions and absolute numbers of the cells are summarized in bar graphs (mean plus SD) (WT: 3 males; Chd8-/-: 3 males). *P < 0.05 vs. WT control group. Fig. 4. Verification of gene expression changes in p53 and mTOR pathways by quantitative real-time RT-PCR. The expression changes of the indicated genes in p53 (A) and mTOR (B) pathways in Chd8-/- Tregs revealed by RNA-seq as shown in Fig 4E were verified by qPCR. The data are normalized to an 18S reference and expressed as mean + SD of triplicates. *P < 0.05, **P < 0.01 vs. WT control group. Fig. 5. Profiles and enrichment plots of ATAC-seq and CHIP-seq A PCA of ATAC-seq. B Fragment size distribution of ATAC-seq. C Profile of ATAC-seq peaks in Chd8-/- (vs WT) Tregs. D GSEA enrichment plots of ATAC up pathways in Chd8-/- Tregs. E PCA of CHIP-seq. F Pie plot of distribution of CHD8 binding peaks in the genome of Tregs. The proportion of peaks in an indicated genomic region was calculated by dividing the number of peaks in that region by the total number of peaks in the genome. The proportion of an indicated genomic region size was determined by dividing the region’s size by the total genome size. The proportion of peaks was then normalized to the proportion of genomic region size. Normalized proportion of peaks is shown. G Profile of ATAC up and ATAC down peaks at CHD8 binding sites in Chd8-/- (vs WT) Tregs. H Profile of ATAC up and ATAC down peaks at non-CHD8 binding sites in Chd8-/- (vs WT) Tregs. Fig. 6. Treg-specific and -nonspecific patterns of CHD8 in DNA binding and regulation of chromatin accessibility and gene expression. A Overlapping of CHD8 binding genes between Tregs and HSPCs. B Overlapping of ATAC up or ATAC down genes between Chd8-/- Tregs and Chd8-/- HSPCs. Cutoff: Log2FC = 0.4. C Overlapping of RNA up or RNA down genes between Chd8-/- Tregs and Chd8-/- HSPCs. Cutoff: Log2FC = 1.0. D Hallmark pathways of CHD8 binding genes in HSPCs. E Hallmark pathways of genes with altered ATAC peaks in Chd8-/- versus WT HSPCs. F Hallmark pathways of genes with altered RNA expression in Chd8-/- versus WT HSPCs.