Fig 2: DNA methylation regulates SDHB expression.a CpG methylation levels in the SDHB promoter region of villi from individuals with RSA and normal controls (n = 7 persons/group). Exact P values in a are listed below: [-1077] 0.000624, [-1135] 2.5 × 10-6, [-1165] 7.6 × 10-7, [-1172] 9.9 × 10-7, [-1185] 0.000193, [-1291] 0.00272, [-1315] 0.00234, [-1421] 9.6 × 10-5, and [-1465] 0.0625. Other CpG sites besides those presented here are shown in Supplementary Fig. 2c. b SDHB promoter CpG methylation levels in villi from normal controls in different trimesters (n = 7 persons of the first trimester; n = 3 persons of the second trimester; n = 3 persons of the third trimester). Exact P values in b are listed below: [-1077] 0.00489, [-1135] 2.2 × 10-5, [-1165] 2.7 × 10-5, [-1172] 2.2 × 10-5, [-1185] 0.000316, [-1291] 0.00575, [-1315] 0.00011, [-1421] 0.000128 and [-1465] 0.00521. Other CpG sites besides those presented here are shown in Supplementary Fig. 2d. c, d CpG methylation levels in JEG3 and HTR8 cells treated with decitabine (c) (n = 3 independent experiments) or azacitidine (d) (n = 3 independent experiments). Exact P values in c are listed below: JEG3: [-1135] 0.00158, [-1165] 0.00463, and [-1172] 0.000308; HTR8: [-1135] 0.000877, [-1165] 0.000448, and [-1172] 0.000206. Exact P values in d are listed below: JEG3: [-1135] 0.000164, [-1165] 3.8 × 10-5, and [-1172] 7.4 × 10-5; HTR8: [-1135] 0.000202, [-1165] 1.0 × 10-5, and [-1172] 0.000112. e, f Gene expression in JEG3 and HTR8 cells treated with decitabine (e) (n = 3 independent experiments) or azacitidine (f) (n = 3 independent experiments). Exact P values in e are listed below: JEG3: SDHB 0.00377, ENO1 0.181, TKT 0.0112, GAPDH 0.339, CS 0.0436, and MDH2 0.973; HTR8: SDHB 0.00112, ENO1 0.559, TKT 0.00433, GAPDH 0.933, CS 0.283, and MDH2 0.763. Exact P values in f are listed below: JEG3: SDHB 0.000527, ENO1 0.171, TKT 0.0362, GAPDH 0.204, CS 0.303, and MDH2 0.398; HTR8: SDHB 0.000527, ENO1 0.383, TKT 0.00184, GAPDH 0.349, CS 0.271, and MDH2 0.638. g SDHB protein levels in cells treated with decitabine or azacitidine. h, i SDHB mRNA (h) (n = 3 independent experiments) and protein levels (i) in cells with knockdown of each methyl-CpG-binding protein. The knockdown efficiencies for each siRNA are shown in Supplementary Fig. 3b, c. j Binding abilities between MBD1 and double-strand DNA in the -1165/-1172 or -1135 CpG sites in EMSA. k Binding ability between c-Fos and double-strand DNA in the -1165/-1135 CpG sites in EMSA. l ChIP followed by PCR showing c-Fos and MBD1 occupancy at the SDHB promoter in cultured JEG3 cells. m ChIP-qPCR shows c-Fos occupancy at the SDHB promoter in MBD1-knockdown cells and control cells (n = 5 independent experiments). n ChIP followed by PCR/qPCR shows c-Fos and MBD1 occupancy at the SDHB promoter in villi from individuals with RSA (n = 3 biologically independent samples) and normal controls (n = 3 biologically independent samples). The left panel shows the ChIP-qPCR results. The right panel shows a representative result of ChIP-PCR. a–f, h, m, n Data represent the mean ± standard error, *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant. Two-tailed unpaired Student’s t tests in a, c–f, h, m, n; one-way ANOVA in b. Source data are provided as a Source Data file.

Fig 3: circNFKB1 associates with ENO1 in human chondrocyte nuclei and silencing ENO1 inhibits IL-1ß-induced ECM degradation.a Schematic illustration of circNFKB1-associated proteins detected by RNA pulldown and MS analysis in human chondrocytes. b Silver staining of RNA pulldown assay with circNFKB1 and control probes. AS antisense. c Mass spectrometry analysis of circNFKB1-binding proteins after RNA pulldown assay. d Western blotting of the interaction between ENO1 circNFKB1 after RNA pulldown. e Confocal images showing the colocalization of circNFKB1 and ENO1 in chondrocytes treated with PBS or IL-1ß. DAPI, 4',6-diamidino-2-phenylindole. Scale bar, 10 µm. f qRT-PCR analysis showing ENO1 mRNA levels after ENO1 knockdown with siRNAs in HCs. g Western blotting analysis showing ENO1 protein levels after ENO1 knockdown. h Representative immunofluorescence images of Aggrecan and Collagen II protein levels in chondrocytes after ENO1 knockdown. Scale bars, 25 µm. i Western blotting analysis of MMP3 and MMP13 expression in ENO1 knockdown chondrocytes. f–i, mean values ± SD are shown, and p values were calculated by two-tailed one-way ANOVA followed by Sidak’s multiple comparison test. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant. b, d, e, and h, representative images are shown.

Fig 4: KMT2D Mutated Tissues Exhibit Aberrant Activation of Glycolysis(A) Top five Gene Ontology (GO) terms for upregulated genes (FDR < 0.05, FC > 2) between KMT2D mutant murine cells and KMT2D WT cells by total RNA-seq analysis.(B) Enrichment plot for HALLMARK glycolysis pathway in all differentially expressed genes (FDR < 0.05, FC > 2) between KMT2D mutant murine cells and KMT2D WT cells by total RNA-seq analysis. Each black bar represents a gene in the pathway.(C) GSEA plot of the HALLMARK glycolysis pathway in differentially expressed genes between human primary melanomas with low versus high KMT2D expression (n = 10 for each group) from the TCGA-SKCM cohort. Each black bar represents a gene in the pathway.(D) GSEA of different MSigDB energy metabolism pathways in differentially expressed genes between KMT2D mutant (carrying truncation, frameshift and post4700aa missense) and WT human tumors from six TCGA tumor groups where KMT2D mutant tumors are n > 10.(E–G) Bar graph showing the relative expression pattern (n = 3 for each sample) of 12 glycolysis enzyme genes (compared to 28S) in KMT2D mutant and WT murine (E) and human (F) cells (details of the system in Figure 2H) as well as in Mut-m1 and Mut-h1 cells with dox-inducible rescue of full-length WT KMT2D expression (G). *p < 0.05, **p < 0.001, ***p < 0.0001. Unpaired t test p values were calculated for each mutant sample versus both WT samples separately, and a higher p value is shown in the figure when both were significant.(H) Immunohistochemistry images demonstrating expression of ENO1, PGK1, and PGAM1, encoded by three glycolysis genes, in iBIP;KMT2D+/+ and iBIP;KMT2DL/L melanoma tumors. Scale bars represent 20 µm.(I–K) Graph showing measurement of glucose uptake and lactate production (n = 3 for each sample) in KMT2D mutant and WT murine (I) and human (J) cells (details of the system in Figure 2H) as well as in the KMT2D mutant mouse Mut-m1 and human Mut-h1 cells with dox-inducible rescue of full-length WT KMT2D expression (K).(L) Bar graph showing relative levels (n = 3 for each sample) of various metabolite intermediates produced during the glycolysis pathway, as measured by selected reaction monitoring tandem mass spectrometry. *p < 0.05, **p < 0.01, ***p < 0.001.(M) Schematic of the glycolysis pathway showing aberrantly activated glycolysis enzymes (in red) and metabolites (in blue) of KMT2D mutant compared to WT conditions.In (E)–(G) and (I)–(L), data are presented as the mean ± SEM (error bars) of at least three independent experiments or biological replicates.

Fig 5: POU6F1 coordinates with RORA to inhibit LUAD progression.A–D Dual-luciferase assay indicating the relative promoter activity of HIF1A, ENO1, PDK1, and PRKCB in A549 and NCI-H1299 cells transfected with mock, RORA, CRISPRi-Scb, or CRISPRi-POU6F1 #1. E, F Real-time qRT-PCR and western blotting assays showing the expression of POU6F1, RORA, HIF1A, ENO1, PDK1, and PRKCB in A549 cells transfected with mock, RORA, CRISPRi-Scb, or CRISPRi-POU6F1 #1. G, H Representative images (left panel) and quantification (right panel) of soft-agar (G) and transwell (H) assays indicating the growth and invasion of A549 cells transfected with mock, RORA, CRISPRi-Scb, or CRISPRi-POU6F1 #1. Student’s t-test and ANOVA compared the difference in A–E and G–H. *P < 0.05, **P < 0.01.