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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.

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Transcriptome analysis of MEFs in OS + chem + VA5 + Dnmt1-KD condition.(a) The ratios of number of upregulated or downregulated genes in MEFs 4 days after OS + chem + VA5 + Dnmt1-KD treatment compared with in control MEFs. Genes whose expression was changed at least two-fold (p < 0.001) were analyzed. (b) The ratios of upregulated genes annotated with particular GO terms. (c) The ratios of downregulated genes annotated with particular GO terms. (d) Relationship of upregulated genes in E13.5 male PGCs and in the treated MEFs compared with control MEFs. (e) GO analysis in commonly upregulated genes in E13.5 male PGCs and in the treated MEFs compared with control MEFs. The GO terms with corrected p-value under 0.05 are shown. (f) Principal Component Analysis (PCA) of control MEFs (MEF), MEFs with OS or OCKS + chem + VA5 + Dnmt1-KD treatments after 4 days in culture, E13.5 male (♂) PGCs, E15.5 female (♀) PGCs, control ESCs, and Max KD ESCs. X-Axis: Component 1 (46.49%), Y-Axis: Component 2 (26.37%), Z-Axis: Component 3 (11.12%). The array data obtained from three biological replicates.
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f4: Transcriptome analysis of MEFs in OS + chem + VA5 + Dnmt1-KD condition.(a) The ratios of number of upregulated or downregulated genes in MEFs 4 days after OS + chem + VA5 + Dnmt1-KD treatment compared with in control MEFs. Genes whose expression was changed at least two-fold (p < 0.001) were analyzed. (b) The ratios of upregulated genes annotated with particular GO terms. (c) The ratios of downregulated genes annotated with particular GO terms. (d) Relationship of upregulated genes in E13.5 male PGCs and in the treated MEFs compared with control MEFs. (e) GO analysis in commonly upregulated genes in E13.5 male PGCs and in the treated MEFs compared with control MEFs. The GO terms with corrected p-value under 0.05 are shown. (f) Principal Component Analysis (PCA) of control MEFs (MEF), MEFs with OS or OCKS + chem + VA5 + Dnmt1-KD treatments after 4 days in culture, E13.5 male (♂) PGCs, E15.5 female (♀) PGCs, control ESCs, and Max KD ESCs. X-Axis: Component 1 (46.49%), Y-Axis: Component 2 (26.37%), Z-Axis: Component 3 (11.12%). The array data obtained from three biological replicates.

Mentions: To understand genome-wide changes in gene expression following OCKS or OS  + chem + VA5 + Dnmt1-KD treatments, we compared the transcriptome of treated MEFs with that of control MEFs. The results indicated that 13% and 16% of genes were upregulated or downregulated, respectively, following OS  + chem + VA5 + Dnmt1-KD treatments (Fig. 4a). Similar results were also obtained following treatment with the OCKS construct that encoded four reprogramming genes (Supplementary Fig. S8a). The upregulated genes in the treated MEFs included not only germ cell-specific genes, but also tissue-specific genes such as nervous system- and immune system-specific genes, as well as a number of stimulus-inducible genes (Fig. 4b; Supplementary Fig. S8b). In the case of OCKS-treated cells, metabolic process-related genes were also upregulated (Supplementary Fig. S8b). The downregulated genes in the treated MEFs included genes related to developmental processes or to metabolic processes (Fig. 4c; Supplementary Fig. S8c).


Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment
Transcriptome analysis of MEFs in OS + chem + VA5 + Dnmt1-KD condition.(a) The ratios of number of upregulated or downregulated genes in MEFs 4 days after OS + chem + VA5 + Dnmt1-KD treatment compared with in control MEFs. Genes whose expression was changed at least two-fold (p < 0.001) were analyzed. (b) The ratios of upregulated genes annotated with particular GO terms. (c) The ratios of downregulated genes annotated with particular GO terms. (d) Relationship of upregulated genes in E13.5 male PGCs and in the treated MEFs compared with control MEFs. (e) GO analysis in commonly upregulated genes in E13.5 male PGCs and in the treated MEFs compared with control MEFs. The GO terms with corrected p-value under 0.05 are shown. (f) Principal Component Analysis (PCA) of control MEFs (MEF), MEFs with OS or OCKS + chem + VA5 + Dnmt1-KD treatments after 4 days in culture, E13.5 male (♂) PGCs, E15.5 female (♀) PGCs, control ESCs, and Max KD ESCs. X-Axis: Component 1 (46.49%), Y-Axis: Component 2 (26.37%), Z-Axis: Component 3 (11.12%). The array data obtained from three biological replicates.
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f4: Transcriptome analysis of MEFs in OS + chem + VA5 + Dnmt1-KD condition.(a) The ratios of number of upregulated or downregulated genes in MEFs 4 days after OS + chem + VA5 + Dnmt1-KD treatment compared with in control MEFs. Genes whose expression was changed at least two-fold (p < 0.001) were analyzed. (b) The ratios of upregulated genes annotated with particular GO terms. (c) The ratios of downregulated genes annotated with particular GO terms. (d) Relationship of upregulated genes in E13.5 male PGCs and in the treated MEFs compared with control MEFs. (e) GO analysis in commonly upregulated genes in E13.5 male PGCs and in the treated MEFs compared with control MEFs. The GO terms with corrected p-value under 0.05 are shown. (f) Principal Component Analysis (PCA) of control MEFs (MEF), MEFs with OS or OCKS + chem + VA5 + Dnmt1-KD treatments after 4 days in culture, E13.5 male (♂) PGCs, E15.5 female (♀) PGCs, control ESCs, and Max KD ESCs. X-Axis: Component 1 (46.49%), Y-Axis: Component 2 (26.37%), Z-Axis: Component 3 (11.12%). The array data obtained from three biological replicates.
Mentions: To understand genome-wide changes in gene expression following OCKS or OS  + chem + VA5 + Dnmt1-KD treatments, we compared the transcriptome of treated MEFs with that of control MEFs. The results indicated that 13% and 16% of genes were upregulated or downregulated, respectively, following OS  + chem + VA5 + Dnmt1-KD treatments (Fig. 4a). Similar results were also obtained following treatment with the OCKS construct that encoded four reprogramming genes (Supplementary Fig. S8a). The upregulated genes in the treated MEFs included not only germ cell-specific genes, but also tissue-specific genes such as nervous system- and immune system-specific genes, as well as a number of stimulus-inducible genes (Fig. 4b; Supplementary Fig. S8b). In the case of OCKS-treated cells, metabolic process-related genes were also upregulated (Supplementary Fig. S8b). The downregulated genes in the treated MEFs included genes related to developmental processes or to metabolic processes (Fig. 4c; Supplementary Fig. S8c).

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