Fig 2: RNA sequencing analysis of cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) isolated from ß-cat loss-of-function (LOF) mice.(A) Volcano plot analysis of mRNA expression for cTECs (left) and mTECs (right) isolated from control (Ctrl) mice and ß-cat LOF mice at 2 wk. Detected genes are plotted as log2 fold change (Ctrl/ß-cat LOF) versus -log10 false discovery rate (FDR) p value. Bold horizontal lines in the plot show the p value of 0.05. (B) Correlation plot analysis for the genes differently altered (p < 0.05) in ß-cat LOF cTECs (left) and ß-cat LOF mTECs (right). Log2 fold changes of genes altered between Ctrl cTECs and ß-cat LOF cTECs are plotted against log2 fold changes of genes altered between Ctrl mTECs and ß-cat LOF mTECs. (C) Quantitative RT-PCR analysis of Cdkn1a mRNA expression normalized to Gapdh levels in UEA1-Ly51+ cTECs isolated from the thymus of control mice and ß-cat LOF mice at 2 wk (means and standard error of the means [SEMs], n = 4). *p < 0.05.

Fig 3: Thymic in situ expression of miR-449/34 sponge reduced mature mTECs. (A) Immunofluoresence staining of thymus sections after in situ injection of control-GFP virus or miR-449/34 sponge-GFP virus with antibodies: K14 (Red) and K8 (Blue), Green indicated GFP expression. (B) Statistic analysis of the K14+GFP+ and K8+GFP+ zone in GFP-expressing area in 3 immunofluoresence images from (A). (C) Flow cytometry analysis of TECs after in situ injection of control-GFP virus or miR-449/34 sponge-GFP virus with antibodies: CD45-PEcy7, Ly51-Alexa 647, MHCII V500. (D) Frequencies of TEC populations (MHCIIhi mTEC: MHCIIhi Ly51-CD45-, MHClow mTEC: MHCIIlow Ly51-CD45-, cTEC: MHCII+ Ly51+CD45-) in (C). Data represent three independent experiments with at least three mice per group. *p < 0.05.

Fig 4: Decreased chromatin accessibility of Pou2f3-regulated genes was consistent with the decreased acetylation regulated by the SIRT6-HDAC9 axis.a Glycolysis-, NF-kappaB pathway-, and PPAR? pathway-associated genes were compared between WT and Sirt6 cKO mTEC bulk RNA-seq by heatmap. The color scale indicates the level of gene expression. b Gene set module scores of the histone deacetylase gene family, sirtuin gene family, and histone lysine acetyltransferase gene family were compared between WT (green) and Sirt6 cKO mouse (red) mTEC populations. c Differential expression of the histone deacetylase gene family, sirtuin gene family, and histone lysine acetyltransferase gene family was calculated by DEseq2 using WT and Sirt6 cKO mTEC bulk RNA-seq. The x-axis represents Log2FC, and the y-axis represents -log10 (P value). The dot size is consistent with the |Log2FC| value. Histone deacetylase-related genes are labeled in bold, and histone acetyltransferase-related genes are not labeled in bold. d Chromatin accessibility peaks around the promoter of the Hdac9 gene between WT and Sirt6 cKO thymic tuft cells are shown in aggregated scATAC-seq tracks. The arrow indicates the length and direction of the peak around the promoter region of Hdac9 detected by scATAC-seq. e Capture of the UCSC (GRCh37/hg19) genome browser showing the human HDAC9 locus in ChIP-seq. f Capture of the UCSC (GRCh37/hg19) genome browser showing the human HDAC9 locus in ATAC-seq. g Western blot analysis of protein acetylation in mTECs (CD45-CD326+UEA-1+Ly51-) isolated from wild-type or Sirt6 cKO mice. h Representative flow cytometric profiles of H3K9ac, H3K18ac, and H3K27ac expressed in CD80- mTECs obtained from 4-week-old Sirt6 cKO mice and littermates. i Frequencies of H3K9ac, H3K18ac, and H3K27ac expression in CD80- mTECs of 4-week-old Sirt6 cKO mice and littermates (n = 4 per group). ***p < 0.001 compared with the identical WT control mice (Student’s t-test). The error bar represents one standard deviation.

Fig 5: Characteristics of thymic epithelial cells (TECs) in postnatal ß-cat loss-of-function (LOF) mice.(A) Intracellular staining of ß-catenin in UEA1-Ly51+ cortical thymic epithelial cells (cTECs; left) and UEA1+Ly51- medullary thymic epithelial cells (mTECs; right) from control mice and ß-cat LOF mice at 2 wk. Histograms show ß-catenin expression in cTECs and mTECs from control mice (blue line) and ß-cat LOF mice (red line). Shaded area and black line represent the fluorescence in the absence of anti-ß-catenin antibody in control TECs and ß-cat LOF TECs, respectively. Plots show net MFI values for ß-catenin in cTECs and mTECs (means and standard error of the means [SEMs], n = 3). The numbers in parentheses indicate percentage of control value. (B) Quantitative RT-PCR analysis of mRNA expression levels (means and SEMs, n = 5) of indicated genes relative to Gapdh levels in UEA1-Ly51+ cTECs (top) and UEA1+Ly51- mTECs (bottom) in the thymus of control mice and ß-cat LOF mice at 2 wk. (C) Flow cytometric analysis of enzyme-digested thymic cells from control mice and ß-cat LOF mice at 2 wk. Shown are representative profiles of EpCAM and CD45 expression in PI- viable cells (left) and UEA1 reactivity and Ly51 expression in CD45-EpCAM+ viable cells (right). The numbers in dot plots indicate the frequency of cells within indicated area. (D) Plots show the number (means and SEMs, n = 6) of cTECs and mTECs in the thymus from control mice and ß-cat LOF mice at 2 wk. (E) Immunofluorescence analysis of ß5t (green), CCL21 (red), and Aire (cyan) on transverse sections of thymus from control mice and ß-cat LOF mice at 2 wk. Representative data from three independent experiments are shown. Bar: 100 µm. Ctrl: Control, LOF: ß-cat LOF. *p < 0.05; **p < 0.01; ***p < 0.001; N.S., not significant.