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. 2021 Apr 12;118(16):e2022760118. doi: 10.1073/pnas.2022760118

Fig. 3.

Fig. 3.

Differentially methylated regions are associated with differentially expressed genes. (A) Heatmap from single-cell RNA sequencing represents the 47 differentially expressed genes between Dnmt3aWT and Dnmt3aKO keratinocyte lineage cells that are associated with a Dnmt3a DMR and exhibit an expression fold change >2 with an FDR <0.01. Grayscale bars represent cells from independent samples, two from each genotype. (B) Relationship between gene expression and methylation changes between Dnmt3aKO and Dnmt3aWT keratinocytes. There are 5,968 differentially expressed genes with an FDR <0.01, of these there are 2,181 genes associated with DMRs. This plot represents expression versus methylation of these 2,181 genes. Significant differential expression, with fold change >2 denoted as red dots. Gray dots represent fold change between 1 and 2. (C) IGV views of differentially methylated regions associated with Meis2, which has annotated DMRs in CpG islands, shelves, shores, and gene body. Gene expression of Meis2 changes by −5.77-fold (FDR 5.7*10−249). Dnmt3aKO expresses Meis2 in 172/15,403 (1.11%) of cells compared to Dnmt3aWT 1,106/10,482 (10.6%). (D) Differentially methylated regions associated with Hoxc8, which has DMRs in shores and gene body. Hoxc8 expression changes by −6.72-fold (FDR 2.51*10−300) and is expressed in 234/15,403 (1.52%) Dnmt3aKO cells compared to 1,375/10,482 (13.1%) Dnmt3aWT. (E) Differentially methylated regions associated with Krt7, which has DMRs in shores, shelves, promoter, and gene body. Expression is changed by 7.19-fold in Dnmt3aKO (FDR 9.2*10−182), and cells expressing increase to Dnmt3aKO 2,551/15,403 (16.6%) from Dnmt3aWT 573/10,482 (5.47%). (F) Differentially methylated regions associated with Irx1, which has DMRs in CpG islands, shores, promoter, and gene body. Expression is changed by 2.02-fold (FDR 1.16*10−109) in Dnmt3aKO keratinocyte lineage cells, and cells expressing increases from 2,873/15,403 (18.7%) Dnmt3aKO cells to 885/10,482 (8.44%) Dnmt3aWT. (G) Simple Western demonstrates protein level expression changes in Irx1 and Krt7 from epidermal cells isolated from the indicated Dnmt3aWT and Dnmt3aKO littermates (bracketed). Actin serves as a loading control.