Fig 2: APPBP2 controls whole-body energy metabolism.a, Changes in the PRDM16–APPBP2 interaction in the presence of EHMT1. b, Changes in PRDM16 polyubiquitination in the presence of EHMT1. c, Immunoblot analysis of the indicated proteins in the inguinal WAT of mice aged 12, 24, 48 and 74 weeks. ß-Actin was used as the loading control. n = 3 per group. d, The whole-body OCR of Adipo-Appbp2-KO and littermate control mice on a regular chow diet at 30 °C in response to treatment with CL-316,243. n = 8 for both groups. e, H&E staining of the inguinal WAT (anterior, middle and posterior regions) in Adipo-Appbp2-KO and control mice in d. Scale bars, 210 µm. Representative images from two biologically independent samples per group. LN, lymph node. f, The body weight of mice on an HFD. n = 10 (Adipo-Appbp2-KO) and n = 11 (control). g, Glucose-tolerance test of mice at 9 weeks of feeding on an HFD. h, Insulin-tolerance test of mice at 10 weeks of feeding on an HFD. i, A model of how CUL2–APPBP2 controls PRDM16 protein stability and beige fat biogenesis. The CUL2–APPBP2 E3 ligase complex catalyses polyubiquitination of PRDM16. Inhibition of CUL2–APPBP2 potently extends the protein half-life of PRDM16, leading to the activation of brown/beige fat genes as well as the repression of pro-inflammatory and pro-fibrosis genes in adipocytes. CUL2–APPBP2 expression in adipose tissues increases with age, showing an inverse correlation with the age-associated decline in PRDM16 protein. EHMT1 stabilizes PRDM16 protein by blocking the PRDM16–APPBP2 interaction. Mito., mitochondrial. For a–c, representative results are shown from two independent experiments. Gel source data are presented in Supplementary Fig. 1. For c, d and f–h, data are from biologically independent samples. For d, f, g and h, data are mean ± s.e.m. Two-sided P values were calculated using two-way repeated-measures ANOVA (d and f–h) followed by Fisher’s least significant difference test (f–h). *P < 0.05, **P < 0.01, ***P < 0.001. Exact P values are shown in Supplementary Table 2.Source data

Fig 3: Euchromatic histone methyltransferases (EHMTs) as inversely regulated genes with CXCL10 level in high-risk neuroblastomas and its association with MYCN amplification. (A) Genes correlating with CXCL10 expression in INSS 4 NBs ranked by increasing Pearson correlation coefficients. Epigenetic modifiers from Xu et al30 are highlighted. Red: positively correlated; blue: negatively correlated. (B) As in (A) but showing genes correlating with MYCN expression. (C) Boxplots showing EHMT2 and EHMT1 expression (RNA-seq, log2) in INSS 4 NBs by MYCN status. (D) Boxplots showing EHMT2 and EHMT1 expression (3′mRNA-seq, log2) in human NB cell lines by MYCN status. (E) Western blots for MYCN, EHMT1, EHMT2, EZH1, EZH2 and β-actin in siMYCN or non-targeting non-targeting siRNA (siNT)-treated SK-N-BE and IMR-32 cells. Representative blots of biological replicates (n=4). (F, G) Quantification of experiment described in (E) from biological replicates (n=4). Error bars; SD. (H) and (I) MYCN-binding peaks in the genomic regions of EHMT2 (upper panels) and EZH2 (lower panel) by ChIP-seq. Cell lines and data set as indicated. (J) and (K) EHMT2 and EHMT1 expression in NB cells versus other cancer cells from the CCLE database. Statistics: two-sided unpaired t-tests (C, D, J, K). Two-sided unpaired t-test with logarithmic values (F, G). Boxplots: Boxes indicate second and third quartile. Bars indicate first and fourth quartile. Horizontal line represents median. *p<0.05; **p<0.01; ***p<0.001. Otherwise p values as indicated. NB, neuroblastoma.

Fig 4: EHMT1 and EHMT2 as proviral factors for SARS-CoV-2 infection(A) qPCR analysis of viral RNA.(B) Western blot analysis of the viral N. The expression of the viral protein was evaluated by the band intensity of N, normalized to that of GAPDH. Cell lysates and culture supernatants were harvested 3 days post-infection.(C) Western blot analysis of the inhibitory effect of UNC0642 on histone methylation. A549-hACE2 cells were treated with UNC0642 and harvested the following day. DMSO was used as a negative control.(D) Inhibitory effect of the EHMT1/2-specific inhibitor UNC0642 on SARS-CoV-2 infection. Viral titers were measured 3 days post-inoculation at an MOI of 0.001. Treatments included pre-treatment (1 h before inoculation), co-treatment during (1 h absorption), and post-treatment (after inoculation).(E) qPCR analysis of viral RNA in cells treated post-infection with UNC0642.(F) Western blot analysis of viral N protein in UNC0642-treated cells. The expression of viral protein was evaluated by the band intensity of N, normalized to that of GAPDH. Data are presented as the mean ± SD of three independent experiments. Statistical analysis was performed using Dunnett’s multiple comparison test. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001.

Fig 5: SARS-CoV-2 and innate immune response in proviral factor-knockout cells(A–C) Quantification of IFNβ (A), CXCL10 (B), IL-6, and TNF-α (C) mRNA in TRIM28, TRIM33, EHMT1, or EHMT2 knockout cells at 24 h post-infection. Poly(I:C) (high molecular weight) was transfected as a positive control in control cells, and cells were harvested 24 h post-infection. For TNF-α, human reference cDNA (30 ng equiv. total RNA) was used as a positive control for qPCR detection. Data are presented as the mean ± SD of three independent experiments. N.D., not detected.