Fig 2: WDR5 was transactivated by TCF3 and promoted the expression of miR-17-5p and HOXA11-AS.a Correlation between the mRNA levels of TCF3 and WDR5 in the TCGA STAD specimens (i) and the GEO GSE66229 dataset (ii). b ChIP-qPCR analysis of TCF3 occupancy in the WDR5 promoter in MGC-803 and MKN-45 cells. c The promoter activity of WDR5 was measured by luciferase reporter gene analysis in HEK293T cells cotransfected with vector containing the target promoter upstream of luciferase and TCF3 or empty vector control. The data are presented as the mean ± s.d. d, e RT-qPCR and western blot analysis of WDR5 mRNA (d) and protein levels (e-i, -ii), respectively, in TCF3 knockdown MGC-803 and MKN-45 cells. f Correlations between the expression level of WDR5 and HOXA11-AS (i), miR-17 (ii), or ATF3 (iii) in the TCGA STAD specimens. g, h ChIP-qPCR analysis of WDR5 and H3K4me3 occupancy in the HOXA11-AS promoter (g) and miR-17 promoter (h) in MGC-803 and MKN-45 cells. i Promoter activity of ß-catenin as measured by luciferase reporter assay in HEK293T cells cotransfected with vector containing the ß-catenin promoter upstream of the luciferase gene and either WDR5 or empty vector controls. The data are presented as the mean ± s.d. n = 3. j, k RT-qPCR analysis of HOXA11-AS (j) and miR-17-5p RNA levels (k) in WDR5-knockdown MGC-803 (i) and MKN-45 cells (ii). l A schematic diagram illustrating the proposed molecular mechanisms of ATF3 inhibition in GC cells and its biological role in the progression of GC. *p < 0.05; **p < 0.01 and ***p < 0.001 by Student’s t test compared with the control group.

Fig 3: Deletion Analysis of ELF1e DIVAC Element(A) Hi-C and DIVAC-trap HTISA data for the ELF1 locus, presented as in Figure 3. Red arrow, location of ELF1e element.(B) GFP fluorescence loss (3 weeks of culture) in Ramos clones infected with GFP7 containing no-DIVAC, ELF1e, or truncation mutants of ELF1e. Data are presented as in Figure 5A. Data points outside of the y axis range are in parentheses.(C) Diagram of entire ELF1e element with tracks for NIPBL, H3K27Ac, H3K4me1, H3K4me3, and GRO-seq. Locations of binding motifs for transcription factors (Z, ZEB1; P, PU.1; M, MEF2; E, E2A; B, BCL6; Y, YY1) are marked. The region of ELF1e retained in the mutants in (B) or deleted from the mutants in (D) is indicated with color-coded bars.(D) Analysis as in (B), but using GFP7 vectors in which small regions were deleted from full-length ELF1e. Deleted regions 1–3 are indicated in (C). Data points outside the y axis range are in parentheses.

Fig 4: EZH2 directly regulates TCF3 which regulates p21 transcription in EC cells.A Relative mRNA expression levels of a panel of key transcription factors potentially regulating p21 normalized to GAPDH analyzed by quantitative real-time PCR in scrambled control (Scr) or EZH2 knockdown ISK and KLE cells. Data shown are mean ± SD (n = 3). **P < 0.01 compared with Scr control. B Immunoblots of EZH2 and TCF3 in Scr or PRC2 core subunit (EZH2, EED, and SUZ12) knockdown ISK and KLE cells. GAPDH served as a loading control. C Enrichment of H3K27me3 on TCF3 promoter in Scr or EZH2 knockdown ISK cells detected by ChIP-qPCR (bottom). Diagram shows positions of ChIP primers at the TCF3 promoter (top). Data shown are mean ± SD (n = 3). **P < 0.01, *P < 0.05 compared with indicated control. D Immunoblots of TCF3, p21, and EZH2 in negative control (NC, vector alone) or enforced expression of TCF3 variants (E12 or E47) in ISK and KLE cells. GAPDH served as a loading control. E, F Cellular proliferation were measured by CCK8 (E) and colony formation assay (F) after enforced expression TCF3 variants (E12 or E47) in ISK and KLE cells. **P < 0.01 vs. NC. G Effect of inhibition of EZH2 activity by GSK126 and EPZ-6438 on TCF3 and p21 protein levels in ISK (left) and KLE (right) cells. GAPDH served as a loading control. H Cell proliferation measured by CCK8 assay after GSK126 and EPZ-6438 treatment in ISK and KLE cells. **P < 0.01 vs. Vehicle treatment. I Immunohistochemical (IHC) staining of TCF3 protein in matched human normal and EC tissues (left). Representative micrographs are shown in original magnification (×400). Scale bars, 50 µm. Total IHC score of TCF3 in matched human normal and EC tissues (n = 26). ***P < 0.001 vs. Normal control (right).

Fig 5: MLL-AF4 controls both H3K27Ac and H3K79me3. (A) Nascent RNA purified from SEM (MLL-AF4, red bars) and RCH-ACV (E2A-PBX1, gray bars) and quantified on a NanoString probe array. Average of two replicates. BTG2 is a gene target transcribed at equal levels in both samples and is presented as a sample control. HOXA9 (a canonical MLL-AF4 target gene) and BCL-2 both display reduced transcription in the E2A-PBX1 sample. ***p < 0.001, NanoStriDE sequencing normalized. (B) BCL2 methylation data from the ECOG E2993 ALL trial (n = 215) revealing reduced DNA methylation at BCL-2 in E2A-PBX1 (green) and MLL-AF4 (red) patients. Each point represents one patient or normal sample; the bar is the average value. ***p < 0.001, *p < 0.05. (C–E) ChIP in MLL-AF4 siRNA treated SEM cells (colored) compared with control (black) using antibodies to H3K27Ac, H3K79me3, or DOT1L. Bars represent the averages of three independent experiments; error bars are SD.