Abstract
Cell differentiation is controlled by specific transcription factors. The functions and expression levels of these transcription factors are regulated by epigenetic modifications, such as histone modifications and cytosine methylation of the genome. In tendon tissue, tendon-specific transcription factors have been shown to play functional roles in the regulation of tenocyte differentiation. However, the effects of epigenetic modifications on gene expression and differentiation in tenocytes are unclear. In this study, we investigated the epigenetic regulation of tenocyte differentiation, focusing on the enzymes mediating histone 3 lysine 9 (H3K9) methylation. In primary mouse tenocytes, six H3K9 methyltransferase (H3K9MTase) genes, i.e., G9a, G9a-like protein (GLP), PR domain zinc finger protein 2 (PRDM2), SUV39H1, SUV39H2, and SETDB1/ESET were all expressed, with increased mRNA levels observed during tenocyte differentiation. In mouse embryos, G9a and Prdm2 mRNAs were expressed in tenocyte precursor cells, which were overlapped with or were adjacent to cells expressing a tenocyte-specific marker, tenomodulin. Using tenocytes isolated from G9a-flox/flox mice, we deleted G9a by infecting the cells with Cre-expressing adenoviruses. Proliferation of G9a-null tenocytes was significantly decreased compared with that of control cells infected with GFP-expressing adenoviruses. Moreover, the expression levels of tendon transcription factors gene, i.e., Scleraxis (Scx), Mohawk (Mkx), Egr1, Six1, and Six2 were all suppressed in G9a-null tenocytes. The tendon-related genes Col1a1, tenomodulin, and periostin were also downregulated. Consistent with this, Western blot analysis showed that tenomodulin protein expression was significantly suppressed by G9a deletion. These results suggested that expression of the H3K9MTase G9a was essential for the differentiation and growth of tenocytes and that H3K9MTases may play important roles in tendinogenesis.
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Abbreviations
- H3K9me1:
-
Mono-methylated lysine 9 at histone H3
- H3K9me2:
-
Di-methylated lysine 9 at histone H3
- H3K9MTase:
-
H3K9 methyltransferase
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418_2015_1318_MOESM1_ESM.pdf
Expression of tenomodulin in cultured tenocytes. Images of DAPI stained (blue) (A and C) and merged images of fluorescence signals for anti-tenomodulin immunostaining (green) and DAPI (blue) (B and D). The cells reacted with anti-tenomodulin antibodies(B) and without primary antibodies as negative control (D) are shown. (PDF 289 kb)
418_2015_1318_MOESM2_ESM.pptx
(A) G9a expression in tenocytes isolated from 7-week-old wild-type mice. mRNA expression of G9a, Col1a1, and Tnmd are shown at 3, 7, and 10 days of tenocyte culture. The relative expression levels of each gene were normalized to the expression of the corresponding gene in tenocytes at day 3, which was set at 1. Asterisks indicate significant differences in mRNA expression between two time points (P < 0.05). (B) Expression of tendon-related genes after G9a deletion in tenocytes isolated from 7-week-old G9a(flox/flox) mice. mRNA expression of G9a, Col1a1, and Tnmd are shown at 7 and 10 days of tenocyte culture. Expression levels were compared between Ad/Cre-infected G9a-null adult tenocytes (black column) with Ad/LacZ-infected control tenocytes (white column). The relative expression levels of each gene were normalized to the expression of the corresponding gene in Ad/LacZ-infected tenocytes at day 7, which was set at 1. Asterisks indicate significant differences in mRNA expression between two time points (P < 0.05). (PPTX 68.4 kb)
418_2015_1318_MOESM3_ESM.pdf
In situ hybridization (ISH) and immunohistochemical analysis of the tendon tissue in mouse embryos at E18.5. ISH images of antisense probes (A-E, G, and H) and sense probes (F and I) on adjacent sections are shown using RNA probes for Tnmd (A and B), col1a1 (C), G9a (D, E, and F), and Prdm2 (G, H, and I). (B), (E), and (H) are higher magnification of the boxed area in (A), (D), and (G). Immunohistochemical analysis was also performed to detect cells expressing tenomodulin protein (J, K, and L). Bright field image (J), merged images of fluorescence signals for anti-tenomodulin immunostaining (green) and nuclear staining with DAPI (blue) (K), and DAPI nuclear staining image (L) are shown. (K) corresponds to the boxed area in (J). Cells positive for Tnmd (A), G9a (D), and Prdm2 (G) mRNA are indicated by black arrowheads. Tenomodulin protein expressing cells are indicated by white arrows (K). Bar = 50 μm. (PDF 954 kb)
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Wada, S., Ideno, H., Shimada, A. et al. H3K9MTase G9a is essential for the differentiation and growth of tenocytes in vitro. Histochem Cell Biol 144, 13–20 (2015). https://doi.org/10.1007/s00418-015-1318-2
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DOI: https://doi.org/10.1007/s00418-015-1318-2