Institute of Human Genetics, Georg-August-University Goettingen, Heinrich-Dueker-Weg 12, 37073 Goettingen, Germany.
Mol Hum Reprod. 2011 Mar;17(3):166-74. doi: 10.1093/molehr/gaq085. Epub 2010 Oct 8.
We previously reported the generation of multipotent adult germline stem cells (maGSCs) from spermatogonial stem cells (SSCs) isolated from adult mouse testis. In a later study, we substantiated the pluripotency of maGSCs by demonstrating their close similarity to pluripotent male embryonic stem cells (ESCs) at the epigenetic level of global and gene-specific DNA methylation. Here, we extended the comparative epigenetic analysis of maGSCs and male ESCs by investigating the second main epigenetic modification in mammals, i.e. global and gene-specific modifications of histones (H3K4 trimethylation, H3K9 acetylation, H3K9 trimethylation and H3K27 trimethylation). Using immunofluorescence staining, flow cytometry and western blot analysis, we show that maGSCs are very similar to male ESCs with regard to global levels and nuclear distribution patterns of these modifications. Chromatin immunoprecipitation real-time PCR analysis of these modifications at the gene-specific level further revealed modification patterns of the pluripotency marker genes Oct4, Sox2 and Nanog in maGSCs that are nearly identical to those of male ESCs. These genes were enriched for activating histone modifications including H3K4me3 and H3K9ac and depleted of repressive histone modifications including H3K27me3 and H3K9me3. In addition, Hoxa11, a key regulator of early embryonic development showed the ESC-typical bivalent chromatin conformation with enrichment of both the activating H3K4me3 and the repressive H3K27me3 modification also in maGSCs. Collectively, our results demonstrate that maGSCs also closely resemble ESCs with regard to their chromatin state and further evidence their pluripotent nature.
我们之前报道了从成年小鼠睾丸中分离的精原干细胞（SSC）生成多能成体生殖细胞（maGSCs）。在后来的研究中，我们通过证明 maGSCs 在表观遗传水平上与多能雄性胚胎干细胞（mESC）在全局和基因特异性 DNA 甲基化水平上非常相似，证实了它们的多能性。在这里，我们通过研究哺乳动物的第二种主要表观遗传修饰，即组蛋白（H3K4 三甲基化、H3K9 乙酰化、H3K9 三甲基化和 H3K27 三甲基化）的全局和基因特异性修饰，扩展了对 maGSCs 和 mESC 的比较表观遗传分析。使用免疫荧光染色、流式细胞术和 Western blot 分析，我们表明 maGSCs 在这些修饰的全局水平和核分布模式方面与 mESC 非常相似。这些修饰的基因特异性水平的染色质免疫沉淀实时 PCR 分析进一步揭示了 maGSCs 中多能性标记基因 Oct4、Sox2 和 Nanog 的修饰模式，与 mESC 几乎相同。这些基因富含激活组蛋白修饰，包括 H3K4me3 和 H3K9ac，并缺乏抑制性组蛋白修饰，包括 H3K27me3 和 H3K9me3。此外，Hoxa11 是早期胚胎发育的关键调节因子，在 maGSCs 中也表现出 ESC 典型的双价染色质构象，富含激活的 H3K4me3 和抑制的 H3K27me3 修饰。总之，我们的结果表明，maGSCs 在染色质状态方面也与 ESC 非常相似，进一步证明了它们的多能性。
