Fig. 4.

Dnmt3a-deficient keratinocytes exhibit a proliferative phenotype. (A) UMAP of Dnmt3a^WT and Dnmt3a^KO single-cell RNA sequencing data representing a pair of samples for each indicated genotype, highlighting the population (in red) identified by unbiased clustering as dominated by expression of genes involved in cell cycling (cluster M). (B) Proliferative gene expression in the proliferative cluster (cluster M) versus the aggregate of the remaining keratinocyte linage clusters (clusters A–L). These data represent the aggregate of Dnmt3a^WT and Dnmt3a^KO cells. Each dot is an individual cell. Expression values are represented by least-squares (LS) means. (C) The number of cells expressing the proliferative marker gene Mki67 increases from 298/10,485 (2.8%) cells in Dnmt3a^WT to 1,509/14,815 (10.2%) cells in the Dnmt3a^KO. (D) Single-cell RNA-sequencing expression data of multiple cell cycle relevant genes, comparing the proliferative cells (cluster M) versus the rest (cluster A–L). *FDR <0.01 and ns, not significant. Expression values are represented by LS means. (E) BrdU uptake in vivo in keratinocytes assessed by flow cytometry (Cd45-negative, single-cell epidermal suspension) comparing Dnmt3a^WT (n = 7) and Dnmt3a^KO (n = 5) mice injected 24 h earlier with BrdU. BrdU uptake is significantly higher in Dnmt3a^KO epidermal keratinocytes (5.6% BrdU+ in Dnmt3a^WT vs. 7.5% BrdU+ in Dnmt3a^KO, P = 0.023).