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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.

No MeSH data available.


Dazl protein was significantly increased in MEFs following OS + chem + VA5 + Dnmt1-KD treatment.(a) Immuno-fluorescence staining of MEFs after 4 days in culture with or without chem + VA5 + Dnmt1-KD in the presence of OS using anti-Dazl antibody. The shown data are representative of three independent experiments. Red: anti-Dazl, Blue: DAPI, scale bar: 200 μm. (b) The ratios of Dazl-positive cells in DAPI-positive cells. Numbers of Dazl- or DAPI-positive cells was estimated by image J. Error bars: S. E. of three biological replicates, *p < 0.05.
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f3: Dazl protein was significantly increased in MEFs following OS + chem + VA5 + Dnmt1-KD treatment.(a) Immuno-fluorescence staining of MEFs after 4 days in culture with or without chem + VA5 + Dnmt1-KD in the presence of OS using anti-Dazl antibody. The shown data are representative of three independent experiments. Red: anti-Dazl, Blue: DAPI, scale bar: 200 μm. (b) The ratios of Dazl-positive cells in DAPI-positive cells. Numbers of Dazl- or DAPI-positive cells was estimated by image J. Error bars: S. E. of three biological replicates, *p < 0.05.

Mentions: To estimate the proportion of MEFs that expressed germ-cell specific genes following induction via OCKS or OS  + chem + VA5 + Dnmt1-KD treatments, we used anti-Dazl antibody to immunostain cultured cells. Notably, Dazl protein was detected in about 50% of of the treated MEFs (Fig. 3; Supplementary Fig. S7). Meanwhile only about 4–7% of the intact MEFs as well as of OCKS + Ctrl siRNA treated MEFs showed the expression of Dazl protein (Fig. 3; Supplementary Fig. S7, data not shown). These findings suggested that upregulation of germ cell-specific genes may generally occur in MEFs after OCKS or OS  + chem + VA5 + Dnmt1-KD treatments.


Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment
Dazl protein was significantly increased in MEFs following OS + chem + VA5 + Dnmt1-KD treatment.(a) Immuno-fluorescence staining of MEFs after 4 days in culture with or without chem + VA5 + Dnmt1-KD in the presence of OS using anti-Dazl antibody. The shown data are representative of three independent experiments. Red: anti-Dazl, Blue: DAPI, scale bar: 200 μm. (b) The ratios of Dazl-positive cells in DAPI-positive cells. Numbers of Dazl- or DAPI-positive cells was estimated by image J. Error bars: S. E. of three biological replicates, *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016969&req=5

f3: Dazl protein was significantly increased in MEFs following OS + chem + VA5 + Dnmt1-KD treatment.(a) Immuno-fluorescence staining of MEFs after 4 days in culture with or without chem + VA5 + Dnmt1-KD in the presence of OS using anti-Dazl antibody. The shown data are representative of three independent experiments. Red: anti-Dazl, Blue: DAPI, scale bar: 200 μm. (b) The ratios of Dazl-positive cells in DAPI-positive cells. Numbers of Dazl- or DAPI-positive cells was estimated by image J. Error bars: S. E. of three biological replicates, *p < 0.05.
Mentions: To estimate the proportion of MEFs that expressed germ-cell specific genes following induction via OCKS or OS  + chem + VA5 + Dnmt1-KD treatments, we used anti-Dazl antibody to immunostain cultured cells. Notably, Dazl protein was detected in about 50% of of the treated MEFs (Fig. 3; Supplementary Fig. S7). Meanwhile only about 4–7% of the intact MEFs as well as of OCKS + Ctrl siRNA treated MEFs showed the expression of Dazl protein (Fig. 3; Supplementary Fig. S7, data not shown). These findings suggested that upregulation of germ cell-specific genes may generally occur in MEFs after OCKS or OS  + chem + VA5 + Dnmt1-KD treatments.

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.