Fig 2: Human TBX3 point mutations are loss-of-function.(A) Western blot for V5 from WT H2.35 cells transfected with WT or mutant TBX3. (B) Immunofluorescence for V5 from WT H2.35 cells transfected with WT or mutant TBX3. (C) Relative secretion of Gaussia luciferase from WT H2.35 24 hours after cotransfection with a Gaussia luciferase expression plasmid along with WT or mutant Tbx3 expression plasmid. The significance in C was calculated using a 1-way ANOVA comparing each group to the Tbx3 WT group and corrected with Dunnett’s post hoc test. (D) Representative immunohistochemistry images of V5+ cells in livers of WT C57BL/6 mice 1 week after AAV injection expressing GFP, WT Tbx3, or Tbx3 harboring the I155S or A280S mutation. Scale bars: 500 μm. (E) Triglyceride secretion assay after 2 weeks of WD feeding from mice from D (GFP, n = 12; Tbx3 WT, n = 17; Tbx3 I155S, n = 17; Tbx3 A280S, n = 17). Two mice from the I155S group were excluded because of technical issues. The results displayed are an aggregation of 2 independent experiments. (F) Quantification of the triglyceride secretion rate of mice from D. The results displayed are an aggregation of 2 independent experiments. (G) Liver weight (left), body weight (middle), and liver/body weight ratios of WT C57BL/6 mice injected with AAV expressing GFP, WT Tbx3, or Tbx3 harboring the I155S or A280S mutations fed a WD for 12 weeks. (H) Representative H&E images of mice from G. Scale bars: 500 μm, left panels; 100 μm, right panels. Significance of the difference in plasma triglycerides among all groups at 180 minutes after P407 injection in E was calculated using a 2-way ANOVA with Tukey’s post hoc test. The statistics displayed in E represent significance of each group compared with the Tbx3-WT group. Significance of the difference in triglyceride secretion rates and the difference in liver weight, body weight, and liver/body weight ratio comparing each group to Tbx3 WT in F and G was done using a 1-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 3: Tbx3 deletion protects against MASLD by transcriptionally upregulating VLDL-TG particle secretion.(A) qPCR results for genes involved in de novo lipogenesis from Tbx3-KO or -WT mice fed a WD for 6 months. (B) qPCR for free fatty acid uptake genes in mice from A. (C) qPCR for β-oxidation genes in mice from A. (D) qPCR for PC biosynthesis and VLDL secretion genes in mice from A. (E) qPCR for PC biosynthesis and VLDL secretion genes in Tbx3-WT or -KO mice fed a WD for 4 weeks. (F) qPCR for β-oxidation genes in mice from E. (G) Ratio of M+14 myristoylcarnitine to M+16 palmitoylcarnitine in the liver of Tbx3-KO or -WT mice fed a WD for 2 weeks. (H) Lipoprotein fractionation (left) and total plasma triglyceride concentration (right) from Tbx3-KO mice fed a WD for 4 weeks. For lipoprotein fractionation, plasma was pooled from 4 mice per group. (I) Experimental setup for in vivo VLDL triglyceride secretion assay. (J) Plasma triglyceride levels over time from Tbx3-KO or -WT mice fed a WD for 2 weeks. Tbx3 KO, n = 8; Tbx3 WT, n = 7. (K) Quantification of the triglyceride secretion rate from mice from K. (L) Plasma concentration of total apolipoprotein B (ApoB) 3 hours after poloxamer injection in mice from K. (M) Experimental setup to induce MASLD with a CDA-HFD. (N) Liver weight (left), body weight (middle), and liver/body weight ratio (right) of Tbx3-KO or -WT mice fed a CDA-HFD for 14 weeks. (O) Representative H&E images in mice from N. 61. Significance of the difference in plasma triglycerides at 180 minutes after P407 injection in J was calculated using a 2-way ANOVA with Šidák’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Scale bars: 500 μm, left panels; 100 μm, right panels.

Fig 4: Tbx3 loss exacerbates diet-induced metabolic syndrome.(A) Intraperitoneal glucose tolerance test from Tbx3-KO or -WT mice on WD for 6 months. Tbx3 KO, n = 10; Tbx3 WT, n = 10. (B) Fasting plasma insulin in mice from A. (C) Plasma free fatty acids (FFA) in mice from A. (D) Plasma triglycerides in mice from A. (E) Intraperitoneal glucose tolerance test in Tbx3-KO or -WT mice fed an NC diet for 6 months. Tbx3 KO, n = 8; Tbx3 WT, n = 8. (F) Fasted plasma insulin in mice from E. (G) Plasma FFAs in mice from E. (H) Plasma triglycerides in mice from E. (I) Plasma cholesterol in mice from E. Significance of the glucose tolerance test in A and E was calculated using a 2-way ANOVA with Šidák’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 5: Somatic mutations identified in TBX3 in liver tissues from MASLD patients.(A) Graphic of the MOSAICS screen workflow. (B) β-Score showing sgTbx3 enrichment in WD- versus NC-fed mice after 6 months of selection. (C) Somatic mutations in TBX3 identified from the livers of patients with MASLD. The mutation in blue represents a synonymous mutation. (D) AlphaFold-predicted TBX3 structure (gray) showing somatic mutations (orange) that do not fall within the DNA-binding domain. (E) X-ray crystal structure (PDB ID: 1H6F) of the TBX3 T-box (gray) bound to DNA (teal) showing somatic mutations (orange) with their respective ΔΔG values.