Fig 2: NSD2 knockdown in HESCs leads to reduced H3K36me2 modifications and the corresponding functional changes. (A) Pie chart depicting the genomic distribution of H3K36me2 peaks. (B) Heatmap showing the occupancy for H3K36me2 across the genome in shNSD2 cells and shNC cells. (C) Chromosomal views showing broadly reduced H3K36me2 peaks after NSD2 knockdown. (D) Venn diagram illustrating the overlap of CUT&Tag-Seq peaks in shNC cells. (E) Venn diagram illustrating the overlap of CUT&Tag-Seq peaks in shNSD2 cells. (F) Venn diagram of downregulated genes after knockdown of NSD2 and decreased genes in patients with RIF and the common peaks in (D), (E). (G) GO terms and KEGG pathways for genes in the overlap of genes downregulated after NSD2 knockdown, genes decreased in patients with RIF, and H3K36me2 peaks in shNC cells but not in shNSD2 cells. (H) Connection between genes, GO terms, and Kegg_Cell Cycle. (I) MCM7 and TTK mRNA levels in endometrial stromal cells during the menstrual cycle (GES111976). The details of the staging of the menstrual cycle are described in Supplementary Table 3. M, menstrual phase; EP, early-proliferative; ≤MP, mid-proliferative; LP, late-proliferative; ES, early-secretory; MS, mid-secretory; LS, late-secretory.

Fig 3: NSD2 regulates MCM7 expression through H3K36me2 modifications on promoter regions. (A) IGV presentation of the enrichment of H3K36me2 in shNC and shNSD2 cells on MCM7 promoter regions. (B) ChIP-qPCR validation of the CUT&Tag-Seq in (A). (C) MCM7 is downregulated by knockdown of NSD2. (D, E) Western blot showing that knockdown NSD2 reduces MCM7 protein levels. (F) MCM7 expression decreased in the proliferative endometrium of patients with RIF (n = 10) compared with FER controls (n = 10). (G, H) Protein levels of MCM7 in the proliferative endometrium of patients with RIF (n = 5) and FER controls (n = 5). (I, J) Representative immunohistochemical images depicting the expression of MCM7 in the endometrium from patients with RIF (n = 10) and FER controls (n = 10). The negative control was normal rabbit IgG. GE, glandular epithelium; S, stroma; ns: not statistically different; *P < 0.05; **P < 0.01.

Fig 4: NSD2 knockdown in HESCs leads to downregulated cell cycle-related gene sets. (A) Volcano plot showing differentially expressed genes after knockdown of NSD2. (B) Enriched GO terms and KEGG pathways of DEGs between the siNSD2 group and the siNC group. (C) Enriched GO terms and KEGG pathways of upregulated DEGs between the siNSD2 group and the siNC group. (D) Enriched GO terms and KEGG pathways of downregulated DEGs between the siNSD2 group and the siNC group. (E) GSEA showing genes enriched by NSD2 knockdown in HESCs. (F) GSEA showing genes enriched between patients with RIF and the FER group.

Fig 5: NSD2 expression is decreased in the proliferative-phase endometrium of patients with RIF compared with that of fertile controls (FER). (A) Heatmap of histone methyltransferases (HMT) and histone demethylase (HDM) gene levels in the RIF and FER groups. (B) EZH2 and NSD2 expression levels in endometrial stromal cells during the menstrual cycle (GES111976). The median menstrual cycle is 28 days in length. The details of staging in the menstrual cycle are described in Supplementary Table 3. M, menstrual phase; EP, early-proliferative; MP, mid-proliferative; LP, late-proliferative; ES, early-secretory; MS, mid-secretory; LS, late-secretory. (C) NSD2 mRNA levels in RIF (n = 12) and FER (n = 12) proliferative endometrium as measured using qRT-PCR. (D, E) Protein levels of NSD2 in the proliferative endometrium of patients with RIF (n = 5) and FER controls (n = 5) as measured using western blotting. (F, G) Representative immunohistochemical images depicting the expression of NSD2 in the proliferative endometrium from patients with RIF (n = 10) and FER controls (n = 10). The negative control was normal rabbit IgG. GE, glandular epithelium; S, stroma; *P < 0.05; **P < 0.01.