Fig 2: Elevation of H3K79hcy may cause inhibition of Menin in high HTL. A The binding levels of H3K79hcy with Men1, Kmt2a, and Wdr82 genes in different regions were analyzed using ChIP-qPCR (n = 3). The P values were calculated with unpaired t test. B–C Overexpressed Mars in normal cultured NE4C induced elevation of H3K79hcy and reduction of Menin and H3K4me3 (n = 3). The symbol “-” indicates that the cell was not transfected with any plasmid; “NC” denotes that the cell was transfected with an empty plasmid; “ + ” denotes that the cell was transfected with a plasmid overexpressing the Mars gene. The P values were calculated with one-way ANOVA plus post-hoc test. D ChIP-qPCR assays validated reduced enrichment of Men1 with H3K79hcy in Mars overexpressed cells (n = 3). The P values were calculated with unpaired t test. E Pathogenic mechanism diagram of HHcy or high HTL leading to NTDs: The formation of HHcy or high HTL could be due to deficiency of folate. This condition increases the level of H3K79hcy modifications, thus epigenetically regulating the expression of the Men1 gene, leading to a reduction in Menin protein expression. With the downregulation of Menin, the modification level of H3K4me3 also decreases correspondingly. Such changes further suppress the DDR pathway of P-ATR-CHK1-NER and may potentially participate in the molecular mechanism of NTDs. CBS, Cystathionine Beta-Synthase; H3, Histone 3; P, phosphorylation

Fig 3: Decreased of Menin-H3K4me3 regulates inhibition of DNA damage response pathway in high HTL. A–B Decreased γH2A.X and activation of P-Atr-Chk1 in Men1-overexpressed cells upon 1 mM HTL treatment (n = 3). “NC” denotes that the cell was transfected with an empty plasmid; “ + ” denotes that the cell was transfected with a plasmid overexpressing the Men1 gene. The P values were calculated with one-way ANOVA plus post-hoc test. C ChIP-qPCR analysis of histone H3K4me3 enrichment in different regions of NER pathway genes: Xpa, Ercc8, Ddb1, Cul4a, Usp7, and Lig1. Blank plasmid or Men1 gene overexpression plasmid was transfected into NE4C cells treated with 0 mM HTL and 1 mM HTL, respectively (n = 3). The P values were calculated with one-way ANOVA plus post-hoc test

Fig 4: Decreased of Menin regulates declined H3K4me3 level in HTL-treated NE4C cells. A The top 30 enriched pathways of H3K4me3 dysregulated genes were analyzed by KEGG. The term “rich factor” refers to the ratio of the number of differentially expressed genes located in a particular pathway entry to the total number of annotated genes located in that pathway entry. B H3K4me3 ChIP-seq analysis on NER genes: Xpa, Ercc8, Ddb1, and Cul4a in 0 and 1 mM HTL-treated NE4C cells. C–D WB analysis demonstrated decreased Menin protein expression and H3K4me3 modification in 1 mM HTL-treated NE4C cells (n = 3). The P values were calculated with one-way ANOVA plus post-hoc test. E–F Overexpressed of Men1 in 0 mM and 1 mM HTL-cultured NE4C, respectively, which further induced an increased of H3K4me3 (n = 3). The symbol “-” indicates that the cell was not transfected with any plasmid; “NC” denotes that the cell was transfected with an empty plasmid; “ + ” denotes that the cell was transfected with a plasmid overexpressing the Men1 gene. The P values were calculated with one-way ANOVA plus post-hoc test

Fig 5: Menin and SUZ12 maintain bivalency balance on developmental genes.a, Pie charts of Menin and SUZ12 peaks in the H9 control hESCs mapped to 3′UTR, 5′UTR, distal intergenic, exon, introns and promoter regions. b,c, Differential analysis for binding of H3K4me3 (b) and H3K27me3 (c) counts. Each dot represents one H3K4me3 or H3K27me3 peak. Peaks that are significantly enriched (red, Padj < 0.05, log2FC > 0) and peaks that are significantly depleted (blue, Padj < 0.05, log2FC < 0) in the KO hESCs, compared to H9 control, are indicated. d, Heatmaps show genomic regions with increased (top panel, each row corresponds to a red dot in b left panel) and decreased (bottom panel, each row corresponds to a blue dot in b left panel) H3K4me3 level in MEN1 KO hESCs compared with control hESCs. NT hESCs were used as control for ATAC-seq, and unmodified H9 hESCs were used as control for CUT&RUN. The genomic regions are ordered according to the H3K4me3 level in the control sample. e, GO analysis on the genes annotated to the peaks with increased H3K4me3 level in MEN1 KO hESCs. Top five GO terms are shown in the dot plot. f, Normalized counts for H3K4me3, H3K27me3 and ATAC peaks for the 265 regions with increased H3K4me3 level in MEN1 KO hESCs. g, Heatmaps show genomic regions with increased (top panel, each row corresponds to a red dot in b right panel) and decreased (bottom panel, each row corresponds to a blue dot in b right panel) H3K4me3 level in SUZ12 KO ESCs compared with control hESCs. The genomic regions are ordered according to the H3K4me3 level in the control sample. h, GO analysis on the genes annotated to the peaks with increased H3K4me3 level in SUZ12 KO hESCs. Top five GO terms are shown in the dot plot. i, Normalized counts for H3K4me3, H3K27me3 and ATAC peaks for the 575 regions with increased H3K4me3 level in SUZ12 KO hESCs. f,i, Box-and-whisker plots: the whiskers represent the minima and maxima, the boxes represent the interquartile range and the centre line represents the median. P values were calculated using two-tailed unpaired t-test comparing KO to control.Source data