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Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment

View Article: PubMed Central - PubMed

ABSTRACT

Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs.

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Inhibition of DNA methylation, TGF-β, and H3K27me3 was essential for Dazl, Tex19.1, and Sycp1 induction.(a) The expression of Dazl, Tex19.1 and Sycp1 in MEFs after 4 days in culture following OS + chem + VA5 + Dnmt1-KD treatment and in control MEFs. The expression in control MEFs for Dazl and Tex19.1, or in MEFs in OS + chem + VA5 + Dnmt1-KD condition for Sycp1 was set as 1.0. (b) The effects of removal of each inhibitor from OS + chem + VA5 + Dnmt1-KD condition on Dazl, Tex19.1, and Sycp1 expression after 4 days in culture. The expression in MEFs following OS + chem + VA5 + Dnmt1-KD treatment was set as 1.0. (c) The expression of Dazl, Tex19.1, and Sycp1 in MEFs after 4 days in culture under OS + chem + VA5 + Dnmt1-KD or OS + ALK5i + DZNep + Dnmt1-KD conditions. The expression in MEFs in the OS + chem + VA5 + Dnmt1-KD condition was set as 1.0. The expression of Dazl, Tex19.1 and Sycp1 was quantified by real-time PCR. Error bars: S.E. of three biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001, n.s.: not significantly different (Student’s t-test).
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f5: Inhibition of DNA methylation, TGF-β, and H3K27me3 was essential for Dazl, Tex19.1, and Sycp1 induction.(a) The expression of Dazl, Tex19.1 and Sycp1 in MEFs after 4 days in culture following OS + chem + VA5 + Dnmt1-KD treatment and in control MEFs. The expression in control MEFs for Dazl and Tex19.1, or in MEFs in OS + chem + VA5 + Dnmt1-KD condition for Sycp1 was set as 1.0. (b) The effects of removal of each inhibitor from OS + chem + VA5 + Dnmt1-KD condition on Dazl, Tex19.1, and Sycp1 expression after 4 days in culture. The expression in MEFs following OS + chem + VA5 + Dnmt1-KD treatment was set as 1.0. (c) The expression of Dazl, Tex19.1, and Sycp1 in MEFs after 4 days in culture under OS + chem + VA5 + Dnmt1-KD or OS + ALK5i + DZNep + Dnmt1-KD conditions. The expression in MEFs in the OS + chem + VA5 + Dnmt1-KD condition was set as 1.0. The expression of Dazl, Tex19.1 and Sycp1 was quantified by real-time PCR. Error bars: S.E. of three biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001, n.s.: not significantly different (Student’s t-test).

Mentions: Finally, we investigated the epigenetic regulatory mechanisms responsible for upregulation of meiosis-related genes in MEFs subjected to the OS + chem + VA5 + Dnmt1-KD treatment. We focused on Dazl, Tex19.1, and Sycp1 because they were highly upregulated in the treated MEFs (Fig. 5a; Supplementary Fig. S9; Supplementary Table S3; Supplementary Table S4). Meanwhile, the expression of those genes in the treated MEFs was lower than that in E13.5 PGCs (Supplementary Fig. S10b), indicating that OS  + chem + VA5 + Dnmt1-KD treatment is still not enough to fully up-regulate the germ cell-specific gene expression. We assessed which treatments were essential for induction of Dazl, Tex19.1, and Sycp1 and found that inhibition of DNA methylation, of H3K27me3, and of TGF-β critically affected induction of these genes (Fig. 5b). In addition, only these three inhibitors in combination with OS (OS + ALK5i + DZNep + Dnmt1-KD) were able to induce levels of Dazl, Tex19.1, and Sycp1 expression comparable to the OS  + chem + VA5 + Dnmt1-KD treatment (Fig. 5c). Similar results were also obtained in ALK5i + DZNep + Dnmt1-KD condition without OS (Supplementary Fig. S11), again suggesting that OS expression is not critical for induction of those germ cell-specific genes.


Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment
Inhibition of DNA methylation, TGF-β, and H3K27me3 was essential for Dazl, Tex19.1, and Sycp1 induction.(a) The expression of Dazl, Tex19.1 and Sycp1 in MEFs after 4 days in culture following OS + chem + VA5 + Dnmt1-KD treatment and in control MEFs. The expression in control MEFs for Dazl and Tex19.1, or in MEFs in OS + chem + VA5 + Dnmt1-KD condition for Sycp1 was set as 1.0. (b) The effects of removal of each inhibitor from OS + chem + VA5 + Dnmt1-KD condition on Dazl, Tex19.1, and Sycp1 expression after 4 days in culture. The expression in MEFs following OS + chem + VA5 + Dnmt1-KD treatment was set as 1.0. (c) The expression of Dazl, Tex19.1, and Sycp1 in MEFs after 4 days in culture under OS + chem + VA5 + Dnmt1-KD or OS + ALK5i + DZNep + Dnmt1-KD conditions. The expression in MEFs in the OS + chem + VA5 + Dnmt1-KD condition was set as 1.0. The expression of Dazl, Tex19.1 and Sycp1 was quantified by real-time PCR. Error bars: S.E. of three biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001, n.s.: not significantly different (Student’s t-test).
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Related In: Results  -  Collection

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f5: Inhibition of DNA methylation, TGF-β, and H3K27me3 was essential for Dazl, Tex19.1, and Sycp1 induction.(a) The expression of Dazl, Tex19.1 and Sycp1 in MEFs after 4 days in culture following OS + chem + VA5 + Dnmt1-KD treatment and in control MEFs. The expression in control MEFs for Dazl and Tex19.1, or in MEFs in OS + chem + VA5 + Dnmt1-KD condition for Sycp1 was set as 1.0. (b) The effects of removal of each inhibitor from OS + chem + VA5 + Dnmt1-KD condition on Dazl, Tex19.1, and Sycp1 expression after 4 days in culture. The expression in MEFs following OS + chem + VA5 + Dnmt1-KD treatment was set as 1.0. (c) The expression of Dazl, Tex19.1, and Sycp1 in MEFs after 4 days in culture under OS + chem + VA5 + Dnmt1-KD or OS + ALK5i + DZNep + Dnmt1-KD conditions. The expression in MEFs in the OS + chem + VA5 + Dnmt1-KD condition was set as 1.0. The expression of Dazl, Tex19.1 and Sycp1 was quantified by real-time PCR. Error bars: S.E. of three biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001, n.s.: not significantly different (Student’s t-test).
Mentions: Finally, we investigated the epigenetic regulatory mechanisms responsible for upregulation of meiosis-related genes in MEFs subjected to the OS + chem + VA5 + Dnmt1-KD treatment. We focused on Dazl, Tex19.1, and Sycp1 because they were highly upregulated in the treated MEFs (Fig. 5a; Supplementary Fig. S9; Supplementary Table S3; Supplementary Table S4). Meanwhile, the expression of those genes in the treated MEFs was lower than that in E13.5 PGCs (Supplementary Fig. S10b), indicating that OS  + chem + VA5 + Dnmt1-KD treatment is still not enough to fully up-regulate the germ cell-specific gene expression. We assessed which treatments were essential for induction of Dazl, Tex19.1, and Sycp1 and found that inhibition of DNA methylation, of H3K27me3, and of TGF-β critically affected induction of these genes (Fig. 5b). In addition, only these three inhibitors in combination with OS (OS + ALK5i + DZNep + Dnmt1-KD) were able to induce levels of Dazl, Tex19.1, and Sycp1 expression comparable to the OS  + chem + VA5 + Dnmt1-KD treatment (Fig. 5c). Similar results were also obtained in ALK5i + DZNep + Dnmt1-KD condition without OS (Supplementary Fig. S11), again suggesting that OS expression is not critical for induction of those germ cell-specific genes.

View Article: PubMed Central - PubMed

ABSTRACT

Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs.

No MeSH data available.


Related in: MedlinePlus