在整個動物界中，組織再生的能力存在顯著差異。在哺乳動物物種中，某些組織具有較高的再生潛力，例如小鼠可以再生毛囊。毛囊是研究傷口誘導的再生的細胞和信號轉導機制的良好模型。在成年皮膚中癒合傷口過程中纖維化和疤痕形成，會導致組織功能喪失，稱為修復。傷口中的組織結構的完全恢復，從而具有正常功能的組織，稱為再生。先前的研究表明Wnt信號對於傷口誘導的毛囊新生 (WIHN) 至關重要，還發現WIHN發生在大鼠、兔子和小鼠上。然而，傷口周圍和中心具有不同的傷口癒合結果，只有傷口中心可以再生毛囊 (Ito et al., 2007)。WIHN會先在傷口周圍表達Msx2，隨後在傷口中心表達 (Hughes et al., 2018)。我們的初步研究結果顯示，在傷口周圍和傷口中心之間存在不同的調控，無論是遺傳學上，例如 β-catenin，LEF1以及SHH，還是表觀遺傳學上，例如HDAC1，HDAC2和EZH2。有趣的是，LEF1和HDAC1在傷口周圍相對於中心處的蛋白質表現不同。LEF1有許多亞型 (Arce et al., 2006; Feder et al., 2020) ，這些亞型被CTNNB1和EP300激活，並且LEF1亞型蛋白表現在邊緣和中心不一樣。組蛋白乙醯化和脫乙醯化在表觀遺傳調控中起著關鍵作用，還受轉譯後修飾（PTM）的調控，並且在傷口邊緣相對於中心處顯示出不同的轉譯後修飾。此外，還有研究表明，β-catenin可以通過轉譯後修飾，例如乙醯化或甲基化，來切換β-catenin的功能（Hoffmeyer et al., 2017）。因此，在這項研究中，我們旨在確定LEF1的特定亞型和HDAC1和β-catenin的特定修飾，以控制小鼠和人類全層傷口癒合期間的再生。我們創建了一個1x1 cm2的傷口，並用Western blot比較傷口周圍和中心之間LEF1和HDAC1的蛋白質表現。LEF1的亞型我們將通過特定的引子來判定。我們使用免疫沉澱來鑑定傷口癒合過程中傷口中心的特定HDAC1和β-catenin修飾。我們發現，HDAC1的修飾是磷酸化，可促進HDAC1的酶活性，而乙醯化則與降低HDAC1的活性有關 (Pflum et al., 2001)。HDAC1的磷酸化表現在傷口邊緣而不是傷口中心。然而，傷口中心的HDAC1的乙醯化表達程度高於傷口邊緣的乙醯化表達水平。接下來，我們通過特定的引子來判定LEF1的特定亞型，結果顯示即使LEF1 mRNA的總量在傷口邊緣有較高的表達，但isoform 1 mRNA表達水平在傷口中心較高。這表明在傷口中心表達了更多具有β-catenin結合域的LEF1。我們的目標是表徵與再生有關的全層皮膚傷口中LEF1的特定亞型和HDAC1和β-catenin的特定修飾，以開發新的臨床療法。

In the entire animal kingdom, there are significant differences in the ability of tissue regeneration. In mammalian species, some tissues have high regenerative potential, for example, mice can regenerate hair follicles. Hair follicles are a good model for studying the cellular and signal transduction mechanisms of wound-induced regeneration. Wound healing by fibrosis and scar formation in adult skin leads to loss of function. This is known as repair. Wound healing by complete restoration of tissue structure leads to normal function. This is known as regeneration. Previous studies indicated Wnt signal is important for Wound-Induced Hair follicle Neogenesis (WIHN) (Ito et al., 2007). WIHN occurs in rats, rabbits and mice (Breedis, 1954; Ito et al., 2007). However, the wound margin and center have different wound healing outcomes, only the wound center can regenerate (Ito et al., 2007, Hughes et al., 2018). WIHN requires expression of epidermal muscle segment Msx2 in the wound margin early, and the wound center later (Hughes et al., 2018). Our preliminary data showed there is different regulation between wound margin and wound center, both genetically, for example β-catenin, LEF1 and SHH, and epigenetically, for example HDAC1, HDAC2 and EZH2. Interestingly, LEF1 and HDAC1 exhibited different protein expression patterns in the wound margin versus center. LEF1 has many isoforms (Arce et al., 2006; Feder et al., 2020), which is activated by CTNNB1 and EP300, and the LEF1 isoform ratio changes from margin to center (unpublished data). Histone acetylation by histone acetyltransferases (HATs) and deacetylation by histone deacetylases (HDACs) play a key role in epigenetic regulation. HATs and HDACs are regulated by post-translational modification (PTM). Interestingly, WIHN shows differential PTM in the wound margin versus center (unpublished data). In addition, another study indicated β-catenin can switch functions through PTMs, such as acetylation or methylation (Hoffmeyer et al., 2017). In this study, we aim to identify the specific isoforms of LEF1 and specific HDAC1 and β-catenin modifications that control regeneration during full-thickness wound healing in mice. We created a 1x1 cm2 wound and observed different ratios of LEF1 isoforms and HDAC1 modifications between wound margin and center with Western blotting. The isoforms of LEF1 will be identified by specific primer sequences via PCR. We used immunoprecipitation to identify the specific HDAC1 and β-catenin modifications in the wound center during wound healing. We found HDAC1 PTMs are phosphorylation that promotes enzymatic HDAC1 activity and acetylation which decreases HDAC1 activity (Pflum et al., 2001). Phosphorylation of HDAC1 was expressed in the wound margin, but not in the wound center. However, acetylation of HDAC1 in the wound center was higher than in the wound margin. Next, we characterized the specific isoforms of LEF1 and showed even though total LEF1 mRNA expression level was higher in wound margin but isoform 1 mRNA expression level was higher in wound center. This indicated that the wound center contains more LEF1 with the β-catenin binding domain. Our goal is to characterize the specific isoforms of LEF1 and the specific modifications of HDAC1 and β-catenin in full-thickness skin wounds associated with regeneration in order to develop novel clinical therapies.

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