Functional impact of DNMT3B knockout on DNA methylation, gene expression, and drug sensitivity in M07e cells. (IMAGE)
Caption
(A) Schematic representation of the CRISPR-Cas9-mediated knockout of DNMT3B (DNMT3B KO) in M07e cells. The U6 promoter drives expression of an hDNMT3B-targeting single-guide RNA (sgRNA), while an EFS promoter controls Cas9 expression along with a T2A-linked puromycin resistance cassette for selection. (B) Western blotting analysis confirmed DNMT3B depletion in DNMT3BKO cells. Wild-type (WT) and scramble control cells served as references. β-actin was used as a loading control. (C) Distribution of differentially methylated regions (DMRs) following DNMT3B KO, categorized as hypermethylated (Hyper-DMRs) or hypomethylated (Hypo-DMRs). DMRs were further classified based on their localization within enhancer-bound (red) or enhancer-unbound (blue) regions. (D) Annotation of Hyper-DMRs (top) and Hypo-DMRs (bottom) across chromatin states (E1–E10) in M07e cells. The distribution of DMRs in promoters (yellow) was significantly higher than in gene bodies (green) (two-tailed t-test, p < 0.05), highlighting preferential methylation changes in regulatory regions. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of genes associated with DMRs, showing significant enrichment in signaling pathways, including axon guidance, focal adhesion, MAPK signaling, and PI3K-Akt signaling. Circle size represents the number of genes in each pathway, and color intensity corresponds to statistical significance (–log10 false discovery rate). (F) The volcano plot displaying differentially expressed genes in DNMT3B KO versus WT cells. Key genes with significant changes (p < 0.05) are labeled, including HPSE2, ITGB2, ITGA5, BMP2, DLX1, NCAM1, STXBP6, and FOLR1. The red and blue indicate up-regulated and down-regulated genes, respectively. (G, H) Genome browser tracks of representative Hypo-DMRs at HPSE2 (G) and ITGB2 (H) loci. Tracks show the presence of active histone modifications (H3K4me3, H3K27ac) and CpG islands (CGI). Loss of DNA methylation at these loci correlates with active chromatin features. (I, J) Quantification of DNA methylation levels at HPSE2 (I) and ITGB2 (J) loci in WT and DNMT3B KO cells. DNA methylation levels were significantly reduced in DNMT3B KO cells (p < 0.05 and p < 0.01, respectively). (K) Cell viability assay assessed the sensitivity of WT and DNMT3BKO M07e cells to venetoclax (VEN) treatment. DNMT3B KO cells exhibited increased sensitivity to VEN, indicating a potential epigenetic mechanism affecting drug response.
Credit
Samrat Roy Choudhury, Akhilesh Kaushal, Pritam Biswas, Cory Padilla, Jay F. Sarthy, Arundhati Chavan, Giselle Almeida Gonzalez, Soheil Meshinchi, Jason E. Farrar
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