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. 2009 Aug;10(8):881-6.
doi: 10.1038/embor.2009.102. Epub 2009 Jul 3.

Epigenetic reprogramming during wound healing: loss of polycomb-mediated silencing may enable upregulation of repair genes

Tanya Shaw  1 , Paul Martin
Affiliations

Epigenetic reprogramming during wound healing: loss of polycomb-mediated silencing may enable upregulation of repair genes

Tanya Shaw et al. EMBO Rep. 2009 Aug.

Abstract

Tissue repair is a complex process that requires wound-edge cells to proliferate and migrate, which in turn necessitates induction of a large repair transcriptome. Epigenetic modifications have emerged as crucial regulators of gene expression. Here, we ask whether epigenetic reprogramming might contribute to the concerted induction of repair genes by wound-edge cells. Polycomb group proteins (PcGs) co-operatively silence genes by laying down repressive marks such as histone H3 lysine 27 trimethylation (H3K27me3), which can be removed by specific demethylases. We show that PcGs Eed, Ezh2 and Suz12 are significantly downregulated during murine skin repair, whereas the newly described demethylases Jmjd3 and Utx are markedly upregulated. Correspondingly, we find a striking reduction of repressive H3K27me3 in the wound epidermis. Quantitative chromatin immunoprecipitation studies have revealed that there is less Eed bound to the regulatory regions of two paradigm wound-induced genes, Myc and Egfr, suggesting that loss of polycomb-mediated silencing might contribute to the induction of repair genes.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Polycomb group proteins are downregulated during skin wound healing, whereas their respective histone demethylases are upregulated. (A–C) Quantitative reverse transcription–PCR analysis of (A) Eed, Ezh2 and Suz12 (PcGs) and (B,C) Jmjd3 and Utx (demethylases) in excisional mouse skin wounds (initial injury: 4 mm biopsy punch; sample collection: 6 mm biopsy punch). The results, normalized to 18 s and presented relative to levels in unwounded skin, show mean±s.e.m. of at least three independent experiments (one-way ANOVA P-values indicated; n=3–5). (D–F) Eed immunohistochemistry (IHC; brown staining) in (D) normal unwounded skin indicates abundant expression in the epidermis (dotted white line indicates the epidermal–dermal boundary); which is downregulated in (E) the leading-edge epithelium 1 day post-wounding (black line demarks the epithelium); and (F) the newly formed epithelium overlying the wound 7 days post-wounding (inset: no primary antibody negative control). (G,H) Ezh2 immunohistochemistry in (G) normal unwounded skin indicates abundant expression in the nuclei of epidermal cells (dotted black line indicates the epidermal–dermal boundary), which is downregulated and predominantly cytoplasmic in (H) the leading-edge epithelium 3 days post-wounding (asterisk demarks the wound edge). (I,J) Western blot analysis of Eed and Jmjd3 during the time-course of wound repair, showing transient downregulation and upregulation, respectively. Scale bars: 50 μm (D,E,G); 30 μm (F,H). ANOVA, analysis of variance; D, dermis; E, epidermis; PcGs, polycomb group proteins; WE, wound epithelium.
Figure 2
Figure 2
The PRC2-mediated repressive histone mark is downregulated during skin wound healing. (A) Western blot analysis of H3K27me3 during the time-course of wound repair, showing transient downregulation. (B) Comparison of H3K27me3 levels in whole skin, epidermis and dermis by western blot, showing expression exclusively in the epidermal compartment. (C) Dispase-mediated isolation of epidermis from unwounded or wounded skin (3 days post-wounding) from three biological replicates, followed by western blot analysis, confirmed a wound-induced reduction in epidermal H3K27me3. (B,C) After blotting, membranes were silver stained, and representative bands are shown as loading controls. (D) A H&E-stained histological section of a day 7 wound, illustrating the complete re-epithelialization at this time point post-wounding (the dotted white line demarks the epidermis–dermis boundary over the central wound site). Scale bar, 130 μm. D, dermis; E, epidermis; GT, granulation tissue; H&E, haematoxylin and eosin; H3K27me3, histone H3 lysine 27 trimethylation; S, scab; WE, wound epithelium.
Figure 3
Figure 3
PRC2-mediated repression is decreased in the regulatory regions of important repair genes, myc and egfr, during wound healing. (A) Transfection of mIMCD3 cells with either empty vector (control) or Eed for 24 h, followed by quantitative reverse transcription–PCR analysis of Egfr and Myc, shows that they are both silenced by Eed. The results, normalized to 18 s and presented relative to levels in vector-transfected controls, show the mean±s.e.m. of at least three independent experiments (one-way ANOVA P-values indicated; n=3–5). (B) Quantitative chromatin immunoprecipitation experiments on unwounded compared with wounded skin, comparing the levels of Eed bound to egfr and myc promoter regions on days 1 and 3 post-wounding, respectively, show less Eed binding during repair. Quantitative PCR results are shown. Data are presented as mean±s.e.m. of three independent experiments (t-test P-values indicated; n=3). (C–E) Western blot analysis of Egfr and Myc during the time-course of wound repair and immunohistochemistry (D) showing increased Egfr staining (brown) in day 3 wound-edge epidermis (asterisk demarks the wound edge) compared with normal unwounded skin. Scale bar, 20 μm. ANOVA, analysis of variance; D, dermis; E, epidermis; WE, wound epithelium.
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