Identification of dihydromyricetin as a natural DNA methylation inhibitor with rejuvenating activity in human skin

Abstract

Changes in DNA methylation patterning have been reported to be a key hallmark of aged human skin. The altered DNA methylation patterns are correlated with deregulated gene expression and impaired tissue functionality, leading to the well-known skin aging phenotype. Searching for small molecules, which correct the aged methylation pattern therefore represents a novel and attractive strategy for the identification of anti-aging compounds. DNMT1 maintains epigenetic information by copying methylation patterns from the parental (methylated) strand to the newly synthesized strand after DNA replication. We hypothesized that a modest inhibition of this process promotes the restoration of the ground-state epigenetic pattern, thereby inducing rejuvenating effects. In this study, we screened a library of 1800 natural substances and 640 FDA-approved drugs and identified the well-known antioxidant and anti-inflammatory molecule dihydromyricetin (DHM) as an inhibitor of the DNA methyltransferase DNMT1. DHM is the active ingredient of several plants with medicinal use and showed robust inhibition of DNMT1 in biochemical assays. We also analyzed the effect of DHM in cultivated keratinocytes by array-based methylation profiling and observed a moderate, but significant global hypomethylation effect upon treatment. To further characterize DHM-induced methylation changes, we used published DNA methylation clocks and newly established age predictors to demonstrate that the DHM-induced methylation change is associated with a reduction in the biological age of the cells. Further studies also revealed re-activation of age-dependently hypermethylated and silenced genes in vivo and a reduction in age-dependent epidermal thinning in a 3-dimensional skin model. Our findings thus establish DHM as an epigenetic inhibitor with rejuvenating effects for aged human skin.

Keywords: DNA methylation; DNAm age clock; DNMT1 inhibition; dihydromyricetin; rejuvenation; skin.

FIGURE 1

Identification of dihydromyricetin (DHM) as a DNMT inhibitor. (A) Establishment of a biochemical assay with human recombinant DNMT1. The plot shows dose-response curves for S-adenosyl-homocysteine (SAH) and doxorubicin (Doxo). (B) Scatter plot showing results from two independent screens. (C) Chemical structures of myricetin (Myr) and dihydromyricetin (DHM). (D) Testing of three independent DHM extracts in an independent biochemical DNMT assay (n = 10). *P_adj < 0.01 (one-way ANOVA with Bonferroni correction).

FIGURE 2

DHM inhibits DNA methylation in primary human keratinocytes. (A) Principal component analysis based on 101,067 differentially methylated probes in the complete dataset obtained with primary human keratinocytes from different donors (n = 13). (B) DHM-dependent hypomethylation in primary human keratinocytes. Box plots indicate the distribution of beta values of the 101,067 differentially methylated probes in the complete dataset. *p = 1.4 × 10⁻³³, t-test. (C) DHM-dependent hypomethylation in epigenomic substructures. *P_adj < 6.7 × 10⁻⁴, t-test.

FIGURE 3

No evidence for critical epigenetic changes upon DHM treatment in keratinocytes. (A). Direct pyrosequencing after bisulfite conversion of the LINE-1 sequence covering methylation of 4 representative CpGs. Bar plot represents mean methylation difference of the analyzed LINE-1 sequence between 20 µM DHM and DMSO treatment in % (n = 4). (B) DNA damage assay. Bar plots depicting number of DNA double strand breaks per cell in primary human keratinocytes upon DHM treatment (blue) and positive control H₂O₂ (yellow) compared to the DMSO control (gray). *p < 0.05 (n = 6; 2-way ANOVA). (C) Apoptosis assay. Bar plots depicting caspase 3/7 activity in primary human keratinocytes upon DHM treatment (blue) and positive control staurosporine (orange) compared to the DMSO control (gray). *p < 0.01 (n = 10; one-way ANOVA, Dunnett’s multiple comparison test).

FIGURE 4

DHM reduces DNA methylation age and predicted wrinkle grade, epigenetic predictors of the skin aging phenotype. Association between DNA methylation age and (A) visual age and (B) skin elasticity, adjusting for the influence of the chronological age using a skin specific clock (Bormann et al., 2016). Pearson correlation coefficient (r) and significance (p) are depicted. DHM reduces the DNA methylation age of cultured primary human keratinocytes determined (C) by the Skin & Blood clock (Horvath et al., 2018) and (D) the newly established human epidermis clock. (E) Induced reduction of the predicted wrinkle grade upon DHM treatment determined with the recently established wrinkle predictor (see accompanying manuscript by Bienkowska et al.). Every symbol represents an individual cell line from an independent donor. *p < 0.05 (Wilcoxon rank-sum test).

FIGURE 5

In vivo and functional effects of DHM on skin aging phenotypes. (A) DHM induces the DNA methylation pattern of the young epidermis in vivo. Volcano plot of hypomethylated CpGs upon topically DHM treatment overlapping with hypomethylated CpGs in young (pink) and old (shaded pink) epidermis, respectively (left). Overlap of hypermethylated CpGs after DHM treatment with hypermethylated CpGs in young and old epidermis, respectively (right). (B) Topical DHM treatment induces the reactivation of selected marker genes (blue bars) that are subject to age-related epigenetic silencing in vivo (shaded pink bars). *p < 0.05 (Wilcoxon rank-sum test). # Significant negative correlation (P_adj < 0.05, Pearson correlation with Holm correction) of gene expression with in vivo wrinkle grade and ## being among the top 10% of genes negatively correlated with in vivo wrinkle grade (see Material and Methods, and Supplementary Figure S9 for details). (C) DHM increases epidermis thickness in an organoid skin aging model. Microscopic images of 3D skin models cultivated with primary fibroblasts either form old donors (top left) or from young donors (top middle) and the resulting epidermal thickness measurements (top right). The effect of DHM was analyzed in the same experimental setup and is shown in the lower panels. Vertical turquoise bars: epidermal thickness measurements. Horizontal black bars: 50 μm *p < 0.005 (Wilcoxon rank-sum test). (D) DHM increases the proliferation of fibroblasts incubated with DHM-conditioned medium obtained from keratinocytes. Schematic representation of the crosstalk experiment is depicted on the left panel. The effect of DHM on number of counted fibroblasts is shown in the right panel (n = 6). *p < 0.05 (Wilcoxon’s signed rank test).