NGS read counts.

NGS read counts.</p

De novo DNMT3a increases nuclear localization at wound proximal site.

(A) DNMT3a and DAPI staining of wound proximal (B) Quantification and kinetics of DNMT3a localization (nuclear v/s cytoplasmic) from wound proximal (≤100 mm) skin sections. (It represents quantification of differentiated keratinocytes from the skin sections of 3 separate biological replicates.) (C) DNMT3a and DAPI staining of scratch wounded in vitro differentiated keratinocyte layer [scale = 20 μm]. (Images are representative of 3 biological replicates.) P-values were calculated using 1-way ANOVA with Dunnett’s test (B), *** P ≤ 0.001, ** P≤ 0.01, ns = P > 0.05. Data underlying the graphs can be found in Fig 2B of S1 Raw Data. DNMT3a, DNA methyltransferase 3A.</p

S2 Fig -

(A) Representative image of unwounded/wound-distal skin section stained with DNMT3a, DAPI, and K5. (B’) A model showing the quantification method of DAPI and DNMT3a stain intensities over the line of interest (1, 2) from proliferating and differentiated keratinocytes, followed by (B”) the plots of intensity values (gray unit) (calculated intensities from 4 biological replicates). Staining of in vitro proliferating and differentiated keratinocytes with (C), DNMT3a/DAPI and (D), DNMT3b/DAPI. (E) DNMT3b/DAPI staining of scratch wounded in vitro differentiated keratinocytes. (F) DNMT3a western blot analysis from control and scratch wounded keratinocytes at 8-hour time point (G), DNMT3a, DAPI, and K5 staining of a completely healed mouse skin section [scale = 20 μm]. Data underlying the graphs can be found in S2B Fig of S1 Raw Data. (TIF)</p

Kinetics of caspase-8 promoter methylation and expression.

(A) Levels of caspase-8 mRNA at different time points post-scratch wound (fold change) (n = 4). (B) In vitro ISH of caspase-8 mRNA showing its levels at scratch margins over time [scale = 10 μm]. (C) In vivo ISH of caspase-8 mRNA showing its levels at wound proximal and distal regions over time (dotted line represents basement membrane, Epi = Epidermis, Der = Dermis) [scale = 20 μm]. (D) Bisulphite sequencing of caspase-8 promoter proximal region (265 bp) shows methylation status of 10 individual CpG sites (columns) from 10 cloned PCR products (rows) at various time points post-scratch wound. Percentage value denotes the percent methylation for each group of CpG sites over time (refer S1D Fig for the sequenced region and primer sites, n = 5 with 2 technical replicates). (Data are shown as mean ± SEM, P-values were calculated using 1-way ANOVA with Dunnett’s test and 2-tailed t test (A), *** P ≤ 0.001, ns = P > 0.05). Data underlying the graphs can be found in Fig 1A of S1 Raw Data.</p

Involvement of DNMT3a and histone modification in regulating caspase-8 expression.

(A) ChIP-qPCR analysis to check DNMT3a and DNMT3b occupancy at caspase-8 promoter in control and scratch wounded keratinocytes (n = 3). (B) qPCR analysis of caspase-8 mRNA in scratch wounded keratinocytes, transduced with either scrambled RNA or DNMT3a shRNA (n = 3). (C) DNA methylation status of caspase-8 promoter in scratch wounded keratinocytes, transduced with either scrambled RNA or DNMT3a shRNA. (D) ChIP-qPCR analysis of H3K9ac, H3K4me3, H3K9me3, and H3K27me3 at caspase-8 promoter in control and scratch wounded keratinocytes (n = 3). (E) Effect of DNMT3a down-regulation on in vitro wound healing assay (n = 3). (Data are shown as mean ± SEM, P-values were calculated using 2-tailed t test (A, B, D), * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ns = P > 0.05.) Data underlying the graphs can be found in Fig 3A, 3B, 3D, and 3E of S1 Raw Data. ChIP, chromatin immunop recipitation; DNMT3a, DNA methyltransferase 3A.</p

Methylation-induced transcriptional reprogramming of epidermal keratinocytes from homeostasis to repair.

(A) Heat map of differentially regulated genes in control and scratch wounded keratinocytes. (B) Scratch wound induced transcriptional down-regulation of genes and status of their associated DNA methylation levels. (C) Fold change of transcriptionally up-regulated genes and their associated DNA methylation levels (MeDIP-qPCR, y-axis = fold change compared to control). (D) DNMT3a and caspase-8 staining of control and psoriatic mouse skin (induced through imiquimod treatment), [scale bar = 100 μm]. Data underlying the graphs can be found in Fig 5A–5C of S1 Raw Data. DNMT3a, DNA methyltransferase 3A.</p

Effect of cellular tension on DNMT3a localization and caspase-8 expression.

(A) Effect of EGTA and blebbistatin on the localization of DNMT3a. (B) Effect of scratch wound DNMT3a localization in presence and absence of calyculin-A. (C) Effect of various matrix stiffness on the localization of DNMT3a. (D) Fold change in the levels of caspase-8 mRNA as a result of varios pharmacological and mechanical approaches of tension modulation (n = 4), [scale bar = 20 μm]. (Data are shown as mean  ±  SEM, P-values were calculated using 2-tailed t test (D), * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ns = P > 0.05). Data underlying the graphs can be found in Fig 4D of S1 Raw Data. DNMT3a, DNA methyltransferase 3A.</p

S3 Fig -

(A) qPCR analysis of caspase-8 mRNA in scratch wounded keratinocytes, pre-treated with 5-Aza-2′-deoxycytidine (5A-dC) or DMSO (n = 4) (B), western blot analysis from keratinocytes transduced with scrambled RNA or DNMT3a shRNA (α-Tub = alpha-tubulin) (data are shown as mean ± SEM, P-values were calculated using 2-tailed t test (A), * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ns = P > 0.05). Data underlying the graphs can be found in S3A Fig and S3B Fig of S1 Raw Data. (TIF)</p

Raw data for Figs 1–5 and S1–S5.

(ZIP)</p

Caspase-8 RNA half-life and CpG positions on its promoter proximal region.

(A) Quantification of caspase-8 mRNA to check its half-life post transcriptional block (using Actinomycin-D) (n = 3). (B) In situ hybridization with anti-sense and sense probe of caspase-8 RNA (in vitro) [scale = 10 μm]. (C) In situ hybridization with anti-sense and sense probe of caspase-8 RNA (in vivo) [scale = 20 μm]. (D) Model showing positions of CpG dinucleotide and SP1 binding sites in caspase-8 promoter proximal region. (Data are shown as mean ± SEM, P-values were calculated using 1-way ANOVA with Dunnett’s test (A), * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ns = P > 0.05.) Data underlying the graphs can be found in S1A Fig of S1 Raw Data. (TIF)</p