log2FC heat maps of age-related DEGs.

(a) Volcano maps of DEGs in each dataset. (b) 2 Mus musculus expression microarrays in pancreatic β cells. (c) A representative image showing the heatmap, that on the right shows the top 10 DEGs between the older and younger groups. (d) Heatmaps of the differential gene expression for datasets. Red represents a high level of expression, blue represents a low level of expression, each column represents one sample and each row represents one gene. The specific locations of representative differential genes in the heat map have been marked on the right.

Functional enrichment analysis of aging islet β cells.

(a, b) Enrichment analysis of differentially expressed genes GO and KEGG. The figure shows the GO and KEGG analysis results of mRNA up-regulation and down-regulation, respectively. (c, d) Circos maps show up-regulated and down-regulated differentially expressed genes (asterisks) that are key to GO enrichment in DNA demethylation and cell senescence.

TET2 is upregulated in the occurrence of diabetes.

(a, b) Western blot and analysis of INS-1E cells or MIN6 treated with high glucose (16.7 mM) for 2 days. (c) Western Blot and analysis of isolated islets in mice fed with NCD or HFD for 16 weeks. (d) Western blot analysis of isolated islets of diabetic db/db mice and their hybrid db/+ litters at 10 weeks of age. (e, f) IF sections of the db/+ and db/db mouse pancreas showed TET2 in red and insulin in green. (g) The average density of TET2 in the nucleus was higher than that in the cytoplasm.

In NCD and HFD mice, TET2 KO mice showed improved glucose tolerance due to age-related increased insulin secretion.

(a) IPGTT was performed in TET2 WT and KO mice fed NCD at weeks 8, 16, 32, and 52 to measure blood glucose concentration and area under the curve (AUC) of IPGTT. (b) Plasma insulin levels in TET2 WT and KO mice fed NCD at 16, 32, and 52 weeks. (c) Release of insulin from mouse islets isolated from 16-week-old TET2 WT and KO mice under low or high glucose conditions. (d) Representative IHC images of TET2 expression in the pancreas of WT mice at a set time point. (e) Statistical quantification of IHC images. (f) The IPGTT and AUC of 6-week-old male TET2 KO and WT mice were analyzed after HFD treatment. (g) NCD treated IPGTT and AUC in WT and TET1/TET3 KO mice at 16 weeks. (h) Insulin secretion in WT and TET1/TET3 KO mice was assessed at a specified time. P-values are displayed accurately.

TET2 deletion enhances β-cell signature gene expression by antagonizing β-cell senescence in NCD or HFD mice.

(a) The pancreatic islets of 24-week-old TET2 KO mice were treated with RNA-Seq, which showed up-regulation of β-cell characteristic genes and down-regulation of age-related characteristic genes (n= 3 per group). (b) QRT-PCR to verify the sequencing results. (c) Representative immunofluorescence of Pdx1 (red) or Glut2 (green) and insulin (gray) in islets of WT and KO mice treated with HFD (52 weeks). (d) Representative immunofluorescence of p16(green), IGF1R(yellow), and insulin (red). (e) Percentage of β-Gal staining positive in pancreatic islets of WT and KO mice treated with HFD. (f) Quantification of p16, β-Gal, IGF1R or LaminB1 immunofluorescence.

overexpression of TET2 accelerates the aging of β cells.

(a-c) For MafA, p16 (red) and Pdx1, IGF1R (green). IF statistical quantification of MafA, Pdx1, p16 and IGF1R. (d) β-Gal staining and quantification.

Methylation levels of PTEN promoter region in TET2 deficient INS-1E were analyzed by PBAT-WGBS.

(a) Bar chart showing genome-wide CpG methylation levels for INS-1E wild type and KO. (b) Scatter plots showing a global methylation comparison between wild type and TET KO INS-1E using 100bp plots. Regions with at least 20% difference in absolute methylation levels between TET-KO and wild-type samples were defined as differentially methylated regions: hypermethylated regions and hypomethylated regions. The triangle represents a methylation difference of at least 20% between the wild-type and TET-KO samples. (c) Distribution of individual CpG methylation levels among the genomic elements shown. DHS: DNase I hypersensitive site. (d) Relative enrichment of hypermethylated and hypomethylated regions with different genomic features in INS-1E KO. CGI: CpG Island. (e) Pie chart showing the distribution of hypermethylated DMR in INS-1E KO apparent cells. (f) Gene ontology analysis of all genes with high methylDMR in the KO INS-1E promoter region. (g) Relative quantitative expression of PTEN in TET-KO INS-1E. (h) Methylation characteristics of the 5’region of PTEN gene in INS-1E extracted from PBAT whole genome sequencing data. The gray shaded box indicates the analysis area. Vertical bar above horizontal line (wild type, green; KO, red) indicates the methylation level (0-1) of a single CpG binary (counting two complementary CPGS), and the blue bar below the horizontal line indicates the detected unmethylation of CpG to distinguish it from the undetected CpG sites. (i) Methylation analysis of PTEN promoter in INS-1E by bisulfite-Sanger sequencing. The hollow and black circles represent unmethylated and methylated CpG sites, respectively. (j) TET assisted sodium bisulfite (TAB) sequencing analysis of PTEN promoter region.

Overall level of H4K16ac elimination by overexpression of TET2 rather than TET1 and TET3.

(a) H4K16ac-ChIP-seq signal heat maps of all genes in INS-1E, MIN6, and βTC6 cell lines. TSS, transcription start site. (b) Density maps of standardized MIN6 H4K16ac ChIP-seq signals in INS-1E cells for different chromatin states defined by the Broad Institute ChromHMM project. The colors in the density map convey a shape that is normalized to the maximum signal distribution within a channel. The quantity H4K16ac is marked on the Y-axis. (c) Map showing the distribution of statistical summaries of H4K16ac ChIP-seq signals in INS-1E cells grouped according to expression quantile (Q) distribution. (d) Bar plots showing the abundance of a single H4 peptide (amino acid 4-17) with different acetylation combinations in INS-1E and MIN6 cells as measured by mass spectrometry. (e) H4K16ac ChIP-seq signal heat maps of all genes in the TETs OE sequence. (f) Average normalized H4K16ac ChIP-seq spectra of all genes in the TETs OE sequence. (g) qRT-PCR quantized H4K16ac ChIP signaling at selected sites after OE TET2, TET1, or TET3. GB, the genome. (h) Examples of H4K16ac ChIP-seq tracks in the OE TETs series. (i) Western blot analysis of H4K16ac and H3 levels after OE TET2, TET1 or TET3.

TET2 regulates the PTEN/MOF/H4K16ac signaling pathway to influence β cell function.

(a) Transcriptome heat maps of islets in mice aged 24 weeks (n=3). (b) qRT-PCR analysis in WT and KO islets. (c) Effects of TET2 OE on PTEN, β cell senescence and β cell recognition genes. (d) Effect of shPTEN on TET2 OE induced changes. (e) Expression of H4K16ac and p16 in INS-1E cells overexpressing MOFs. (f) MOF overexpression treatment for 24 h confirmed the effect of TET2 on β cell function through MOF. (g) Expression of p21 and p16 in INS-1E cells with TET2 overexpression or MOF overexpression or double overexpression. (h) Effects of TET2 knockdown on PTEN/MOF signaling pathway and aging markers in INS-1E cells. Ctrl, contrast; KD, knock it down. Effect of TET2 downregulation on insulin secretion under low and high glucose conditions.