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

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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.


Changes of germ cell-specific gene expression in MEFs in OCKS + chem + VA5 + Dnmt1-KD condition.The expression of germ cell-specific genes was quantified by real-time PCR in MEFs after transfection of the expression vector encoding the Yamanaka factors (Oct4, c-Myc, Klf4, Sox2: OCKS) with or without addition of tranylcipromine, BIX-01294, DZNep (chem), VPA, ALK5i (VA5), and Dnmt1 Knocked-Down (KD) (OCKS + chem + VA5 + Dnmt1-KD, or OCKS + Ctrl siRNA) after 2, 3, 4 days in culture. The expression level of each gene in MEFs with OCKS + chem + VA5 + Dnmt1-KD was set as 1.0. Error bars: S.E. of three biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).
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f1: Changes of germ cell-specific gene expression in MEFs in OCKS + chem + VA5 + Dnmt1-KD condition.The expression of germ cell-specific genes was quantified by real-time PCR in MEFs after transfection of the expression vector encoding the Yamanaka factors (Oct4, c-Myc, Klf4, Sox2: OCKS) with or without addition of tranylcipromine, BIX-01294, DZNep (chem), VPA, ALK5i (VA5), and Dnmt1 Knocked-Down (KD) (OCKS + chem + VA5 + Dnmt1-KD, or OCKS + Ctrl siRNA) after 2, 3, 4 days in culture. The expression level of each gene in MEFs with OCKS + chem + VA5 + Dnmt1-KD was set as 1.0. Error bars: S.E. of three biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).

Mentions: Reportedly, inhibition of DNA methylation in mESCs results in induction of germ-cell-specific genes including Dazl and Stella21. Therefore, we tested the OCKS + Max-KD + Atf7ip-KD + VA5 combination or the OCKS + chem + VA5 combination with either of two inhibitors of DNA methylation, Dnmt1-KD or 5-Aza-cytidine (Aza) (Supplementary Fig. S1); the OCKS + chem + VA5 + Dnmt1-KD combination significantly induced Dazl, Stra8, Blimp1, and Stella in addition to Vasa after 2 days in culture (Supplementary Fig. S5a). Moreover, Vasa and Dazl expression was further elevated in a culture period-dependent manner until 4 days (Fig. 1). However, the expression of Vasa tended to decrease after 6 in culture (Supplementary Fig. S6), and the expression from the Vasa::RFP reporter was not detectable after 2 weeks in culture (data not shown). Meanwhile, the OCKS + chem + VA5 + Dnmt1-KD combination did not induce somatic genes such as Hoxa1 and Hoxb1 (Supplementary Fig. 5b); this finding suggested that induction of germ cell-specific genes by OCKS + chem + VA5 + Dnmt1-KD was not due to non-specific transcription activation.


Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment
Changes of germ cell-specific gene expression in MEFs in OCKS + chem + VA5 + Dnmt1-KD condition.The expression of germ cell-specific genes was quantified by real-time PCR in MEFs after transfection of the expression vector encoding the Yamanaka factors (Oct4, c-Myc, Klf4, Sox2: OCKS) with or without addition of tranylcipromine, BIX-01294, DZNep (chem), VPA, ALK5i (VA5), and Dnmt1 Knocked-Down (KD) (OCKS + chem + VA5 + Dnmt1-KD, or OCKS + Ctrl siRNA) after 2, 3, 4 days in culture. The expression level of each gene in MEFs with OCKS + chem + VA5 + Dnmt1-KD was set as 1.0. Error bars: S.E. of three biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).
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Related In: Results  -  Collection

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f1: Changes of germ cell-specific gene expression in MEFs in OCKS + chem + VA5 + Dnmt1-KD condition.The expression of germ cell-specific genes was quantified by real-time PCR in MEFs after transfection of the expression vector encoding the Yamanaka factors (Oct4, c-Myc, Klf4, Sox2: OCKS) with or without addition of tranylcipromine, BIX-01294, DZNep (chem), VPA, ALK5i (VA5), and Dnmt1 Knocked-Down (KD) (OCKS + chem + VA5 + Dnmt1-KD, or OCKS + Ctrl siRNA) after 2, 3, 4 days in culture. The expression level of each gene in MEFs with OCKS + chem + VA5 + Dnmt1-KD was set as 1.0. Error bars: S.E. of three biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test).
Mentions: Reportedly, inhibition of DNA methylation in mESCs results in induction of germ-cell-specific genes including Dazl and Stella21. Therefore, we tested the OCKS + Max-KD + Atf7ip-KD + VA5 combination or the OCKS + chem + VA5 combination with either of two inhibitors of DNA methylation, Dnmt1-KD or 5-Aza-cytidine (Aza) (Supplementary Fig. S1); the OCKS + chem + VA5 + Dnmt1-KD combination significantly induced Dazl, Stra8, Blimp1, and Stella in addition to Vasa after 2 days in culture (Supplementary Fig. S5a). Moreover, Vasa and Dazl expression was further elevated in a culture period-dependent manner until 4 days (Fig. 1). However, the expression of Vasa tended to decrease after 6 in culture (Supplementary Fig. S6), and the expression from the Vasa::RFP reporter was not detectable after 2 weeks in culture (data not shown). Meanwhile, the OCKS + chem + VA5 + Dnmt1-KD combination did not induce somatic genes such as Hoxa1 and Hoxb1 (Supplementary Fig. 5b); this finding suggested that induction of germ cell-specific genes by OCKS + chem + VA5 + Dnmt1-KD was not due to non-specific transcription activation.

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.