Fig. 5. Increased DNAme at Polycomb CGIs impairs differentiation-induced transcriptional activation of target genes.

(A) MA plot comparing log₁₀ of average normalized reads per gene versus log₂ fold change (LFC) in gene expression between DNMT3A1^W330R and DNMT3A1^WT. Significantly differentially expressed genes are highlighted in green (LFC > 2 and q < 0.01) or red (LFC < −2 and q < 0.01) (two biological replicates). (B) Bar plot showing distribution of 10T DNMT3A1^W330R–hypermethylated genes into gene expression deciles in DNMT3A1^WT 10T cells. (C) Bar plot of log₁₀ P values of select GO terms of 10T DNMT3A1^W330R–hypermethylated genes. (D) Top: Schematic of 10T differentiation to adipocytes. Bottom: Bar plot of normalized Red Oil O absorption in day 10 adipocytes. Representative images of Red Oil O staining are shown. (E) MA plot comparing log₁₀ of average normalized reads per gene in DNMT3A1^WT undifferentiated and day 6 adipocyte-differentiated 10T cells versus LFC in gene expression. Significantly differently expressed genes are highlighted in green [LFC > 2 and false discovery rate (FDR) < 0.01] or red (LFC < −2 and FDR < 0.01) (two biological replicates). (F) Gene expression (log₂ RPM) box plot of top 500 up-regulated genes in adipocytes from days 2 to 6 (two biological replicates). Student’s t test, (ns) P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001. (G) Enrichment plot showing the distribution of the percentage of DNAme over the promoters of up-regulated genes during adipogenesis for DNMT3A1^WT, DNMT3A1^W330R, and DNMT3A^W330R+181E undifferentiated 10T cells. (H) Genome browser view of the percentage of DNAme and gene expression of undifferentiated and day 6 adipocyte-differentiated DNMT3A1^WT, DNMT3A1^W330R, and DNMT3A^W330R+181E 10T cells over Lpl. Right: Average DNAme at Lpl promoter.