TET3 regulates terminal cell differentiation at the metabolic level

Abstract

TET-family members play a critical role in cell fate commitment. Indeed, TET3 is essential to postnatal development due to yet unknown reasons. To define TET3 function in cell differentiation, we have profiled the intestinal epithelium at single-cell level from wild-type and Tet3 knockout mice. We have found that Tet3 is mostly expressed in differentiated enterocytes. In the absence of TET3, enterocytes exhibit an aberrant differentiation trajectory and do not acquire a physiological cell identity due to an impairment in oxidative phosphorylation, specifically due to an ATP synthase assembly deficiency. Moreover, spatial metabolomics analysis has revealed that Tet3 knockout enterocytes exhibit an unphysiological metabolic profile when compared with their wild-type counterparts. In contrast, no metabolic differences have been observed between both genotypes in the stem cell compartment where Tet3 is mainly not expressed. Collectively, our findings suggest a mechanism by which TET3 regulates mitochondrial function and, thus, terminal cell differentiation at the metabolic level.

Fig. 1. Enterocytes are the main cell-type expressing Tet3 in the intestinal epithelium.

a, b, c and d Representative images of multiplex RNAscope in situ hybridization for Tet1, Tet2 and Tet3 plus one cell type—specific marker: a Olfm4 (intestinal stem cells), b Chga (enteroendocrine cells), c Muc2 (goblet cells) and d Vil1 (enterocytes) in wild-type intestinal epithelial sections. Scale bars represent from left to right, 100, 20 and 10 μm, respectively. a, b, c and d Co-expression of Tet3 with each cell type—specific marker at the single-cell level in the intestinal epithelium. The axes represent the number of RNA copies detected for each gene by RNAscope in situ hybridization and the color code indicates the number of cells for each combination. The total number of analyzed cells is shown at the top. Source data are provided as a Source Data file.

Fig. 2. Enterocytes acquire an aberrant transcriptional signature in the absence of TET3.

a Cluster color-coded scRNA-seq t-SNE visualization of 5933 E18.5 wild-type and 5983 E18.5 Tet3 knockout small intestinal epithelial cells. Clusters are labelled with numbers and cell identities are delineated with dashed lines. b t-SNE visualizations of (upper panel) only wild-type cells n = 5933 cells and (lower panel) only wild-type specific clusters n = 3816 cells. c t-SNE visualizations of (upper panel) only Tet3 knockout cells n = 5983 cells and (lower panel) only Tet3 knockout specific clusters n = 3211 cells. d Cluster color-coded (upper panel) or genotype color-coded (lower panel) cell differentiation trajectory containing both wild-type (n = 5933) and Tet3 knockout (n = 5983) cells for each genotype. Black and white arrows in lower panel show differentiation into the physiological and aberrant absorptive lineage, respectively. Dashed lines delimit wild-type and Tet3 knockout—specific clusters. e, h Top 8 significantly enriched ontology terms identified using g:Profiler in specific wild-type clusters (n = 3816 cells) or in specific Tet3 knockout clusters (n = 3211 cells), respectively. f, i Expression levels of genes representative of the mitochondrial electron transport pathway or lipid metabolism pathway in specific wild-type clusters (n = 3816 cells) or in specific Tet3 knockout clusters (n = 3211 cells), respectively. Median as well as the first quartile and the third quartile are depicted in green and yellow, respectively. g GSEA plot showing oxidative phosphorylation as a negatively enriched pathway in Tet3 knockout epithelial cells. j Differential expression and statistical significance between wild-type (n = 3816 cells) and Tet3 knockout (n = 3211 cells) specific clusters for mitochondrial subunits showing p-values < 0,05. Fisher’s one-tailed test with g:SCS multiple testing correction (e, h) and Wilcoxon rank-sum test with Benjamini-Hochberg correction (f, i and j) was used.***p-value < 0.001. Source data are provided as a Source Data file.

Fig. 3. Mitochondrial morphology remains immature in the absence of TET3.

a Representative transmission electron microscopy images from E18.5 wild-type and Tet3 knockout mitochondria located at intervilli and villi tips are shown. Semi-thin sections images with boxed areas indicating the analyzed villi regions are included. Scale bars: 500 nm. b Quantitative mitochondrial area comparison between wild-type intervilli, Tet3 knockout intervilli, wild-type villi tip and Tet3 knockout villi tip (n = 45, 53, 155, and 150 mitochondria, respectively). Center line, median; box limits, upper and lower quartile; whiskers, 1.5x interquartile range; points, all measures. Student t test, two-tailed. Source data are provided as a Source Data file.

Fig. 4. TET3 regulates mitochondrial F₁F₀-ATP synthase assembly.

a Blue-Native gel electrophoresis followed by in gel complex I activity or western blot with anti-NDUFA9 in isolated mitochondria from wild-type and Tet3 knockout fibroblasts. SDHA was used as loading control. I, II, III and IV refer to the different mitochondrial complexes associated into supercomplexes. Subscript number refers to the units of each complex. n = 2 biologically independent replicates, each sample corresponds to a pool of two independent biological samples. b Mitochondrial complex I NADH: Fe(CN)6 oxidoreductase (left panel) and NADH:ubiquinone (middle panel) activities spectrophotometrically measured in isolated mitochondria from wild-type and Tet3 knockout fibroblasts. Percentage of complex I activation in isolated mitochondria (right panel). n = 3 biologically independent samples. c Bovine ATP synthase dimer state1:state3 model (PDB ID: 7AJD) in which the subunits showing lower expression levels in the absence of TET3 have been color-coded. d Clear native gel electrophoresis followed by in gel ATPase activity or blue native gel electrophoreses followed by western blot with anti-ATPβ from isolated mitochondria in wild-type and Tet3 knockout fibroblasts. ATP synthase O, oligomer; D, dimer; M, monomer. n = 2 biologically independent replicates, each sample corresponds to a pool of two independent biological samples. e Dimer/monomer ratio quantification detected by in gel activity in (d). n = 2 biologically independent replicates, each sample correspond to a pool of two independent biological samples. f F₁F₀ATP synthase activity measured spectrophotometrically in mitochondria isolated from wild-type and Tet3 knockout fibroblasts. n = 2 biological replicates. g Mitochondrial membrane potential and mitochondrial mass quantification in wild-type and Tet3 knockout fibroblasts, as measured by confocal imaging. n = 3 biologically independent samples. a.u., arbitrary units. h Citrate synthase activity measured spectrophotometrically in wild-type and Tet3 knockout fibroblasts. n = 3 biological replicates. i Oxygen consumption rate (OCR)-derived quantification of basal respiration in wild-type and Tet3 knockout fibroblasts. n = 6 biologically independent replicates. Data are presented as mean values +/− SD. Student’s t test, two-tailed. Source data are provided as a Source Data file.

Fig. 5. TET3 deletion leads to hypermethylation in the intestinal epithelium.

a, b WGBS data represented as 5mC average levels over a different gene regions and b gene body regions for all annotated genes in wild-type and Tet3 knockout intestinal epithelium. The methylation level has been calculated after dividing each region in 20 bins. n = 2 biological replicates. c Distribution of hyper and hypo DMRs across different gene regions. n = 2 biological replicates. d Distribution of hyper and hypo DMRs across different chromosomes. TE, repeat elements. n = 2 biological replicates. e Heatmap of H3K27ac and H3K4me1 at all enhancers identified in P0 intestinal cells. Data from ENCODE. f Line plot of H3K27ac and H3K4me1 enrichment at all enhancers. g Heatmap of H3K27ac, H3K4me1 and DNA methylation from WGBS in wild-type and Tet3 knockout intestinal epithelium at hypermethylated enhancers. h DNA methylation levels at hypermethylated enhancers in Tet3 knockout intestinal epithelial cells. i Percentage of hyperDMRs associated to enhancers in P0 intestinal cells. j, k Top 20 gene ontology terms enriched in all Tet3 knockout hyperDMRs (j) or only in Tet3 knockout hyperDMRs located at the promoter (k). Rich factor indicates the ratio of the number of hypermethylated genes in the pathway to the total number of genes in that pathway.

Fig. 6. TET3 loss-of-function leads to an abnormal metabolic profile.

a Hematoxylin and eosin—stained sections as well as MS-MALDI outlined sections for wild-type and Tet3 knockout. Insets in the intestinal section images show the location of the magnified villus in which the measured regions are indicated. Red and green refer to base and tip, respectively. Scale bars, 200 and 50 µm b–i MALDI-MS relative quantification of metabolites. Center line refers to the median; box limits, upper and lower quartiles; whiskers 1.5x interquartile range; points, outliers. n = 4 independent measurements from four different histological sections. *p-value < 0,05, **p-value < 0,01 and ***p-value < 0,001. Student’s t test, two-tailed. Source data are provided as a Source Data file.

Fig. 7. TET3 regulates terminal cell differentiation at the metabolic level.

Tet3 is almost not expressed at the stem cell compartment, thus both wild-type and Tet3 knockout intestinal stem cells show an identical transcriptional profile and mitochondrial morphology. Upon differentiation, Tet3 expression increases in the enterocytes as they migrate towards the tip of the villi. In the absence of TET3, genes coding for critical ATP synthase subunits exhibit reduced gene expression levels leading to an abnormal mitochondrial morphology and an aberrant metabolic signature specifically in enterocytes located at the tip of the villi, where Tet3 is expressed at its highest level, but not at the base where it is almost not expressed (Fig. 3, Fig. 6 and Fig. 7). Finally, a biochemical analysis in fibroblasts identified an ATP synthase deficiency assembly in the absence of TET3. As Tet3 knockout fibroblasts exhibit: i) a reduction in the expression levels of the same critical ATP synthase subunits affected in Tet3 knockout enterocytes and; ii) a metabolic profile very similar to Tet3 knockout enterocytes, we have extrapolated the ATP synthase impairment observed in fibroblasts to enterocytes.