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Transcriptional Mechanisms of Proneural Factors and REST in Regulating Neuronal Reprogramming of Astrocytes.

Masserdotti G, Gillotin S, Sutor B, Drechsel D, Irmler M, Jørgensen HF, Sass S, Theis FJ, Beckers J, Berninger B, Guillemot F, Götz M - Cell Stem Cell (2015)

Bottom Line: We found that Neurog2 and Ascl1 rapidly elicited distinct neurogenic programs with only a small subset of shared target genes.Notably, in astrocytes refractory to Neurog2 activation, the underlying neurogenic program remained amenable to reprogramming by exogenous NeuroD4.Our findings support a model of temporal hierarchy for cell fate change during neuronal reprogramming.

View Article: PubMed Central - PubMed

Affiliation: Physiological Genomics, Biomedical Center, University of Munich, 80336 Munich, Germany; Institute for Stem Cell Research, Helmholtz Centre Munich, 85764 Neuherberg, Germany.

No MeSH data available.


Related in: MedlinePlus

Deletion of REST Removes Reprogramming Block in Astrocytes(A) Schematic representation of the experimental procedure.(B–E) Micrographs of Neurog2ERT2-infected astrocytes (red) with early (B and C) or late (D and E) deletion of REST by infection with a Cre containing viral vector (green) immunostained for the neuronal marker βIII-tubulin (white) at 8 DPI. Yellow arrowheads indicate triple positive cells (DsRed, YFP, βIII-tubulin) while white arrowheads indicate double positive cells (DsRed, GFP). Scale bars, 150 μm.(F) Histogram depicting the proportion of co-transduced double positive cells (red and green) for the astrocytic marker (GFAP, white bars) or the neuronal marker (βIII-tubulin, black bars). Mean ± SEM, three independent biological samples; two-tailed unpaired t test, ∗p < 0.05; ∗∗∗p < 0.001.(G) Postnatal (day 6–7) mouse cortical astrocytes transduced with Ascl1 or Neurog2 are reprogrammed into neurons. However, when cells are maintained longer in culture, increasing levels of H4K20me3 modify the local chromatin environment that becomes favorable to the repressive complex REST. Consequently, Neurog2 fails to access the NeuroD4 promoter. This is bypassed by common downstream transcription factors to both Ascl1 and Neurog2 that are able to generate neurons also in prolonged astrocytic cultures. Unidentified REST co-factors might be recruited to the locus to further remodel the chromatin over time.See also Figure S7.
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fig7: Deletion of REST Removes Reprogramming Block in Astrocytes(A) Schematic representation of the experimental procedure.(B–E) Micrographs of Neurog2ERT2-infected astrocytes (red) with early (B and C) or late (D and E) deletion of REST by infection with a Cre containing viral vector (green) immunostained for the neuronal marker βIII-tubulin (white) at 8 DPI. Yellow arrowheads indicate triple positive cells (DsRed, YFP, βIII-tubulin) while white arrowheads indicate double positive cells (DsRed, GFP). Scale bars, 150 μm.(F) Histogram depicting the proportion of co-transduced double positive cells (red and green) for the astrocytic marker (GFAP, white bars) or the neuronal marker (βIII-tubulin, black bars). Mean ± SEM, three independent biological samples; two-tailed unpaired t test, ∗p < 0.05; ∗∗∗p < 0.001.(G) Postnatal (day 6–7) mouse cortical astrocytes transduced with Ascl1 or Neurog2 are reprogrammed into neurons. However, when cells are maintained longer in culture, increasing levels of H4K20me3 modify the local chromatin environment that becomes favorable to the repressive complex REST. Consequently, Neurog2 fails to access the NeuroD4 promoter. This is bypassed by common downstream transcription factors to both Ascl1 and Neurog2 that are able to generate neurons also in prolonged astrocytic cultures. Unidentified REST co-factors might be recruited to the locus to further remodel the chromatin over time.See also Figure S7.

Mentions: To examine the effect of REST deletion on Neurog2ERT2-dependent neuronal conversion, RESTflox astrocytes were co-infected with Neurog2ERT2- and Cre-encoding viruses soon after being plated, or with a 5 day delay (Figure 7A). Cultures were then treated for 3 consecutive days with OHT and analyzed 8 DPI (Figures 7B–7E, Figures S7A–S7D). As previously reported (Xue et al., 2013), REST deletion generated a fraction of βIII-tubulin+ cells on its own without Neurog2ERT2 activation (around 20%, Figure 7F); strikingly, however, 90% of Cre/Neurog2ERT2 transduced cells were βIII-tubulin+ after early REST deletion and delayed Neurog2ERT2 activation (Figure 7D). Delayed Cre-mediated REST deletion still allowed 50% of Cre/Neurog2ERT2 double positive cells to convert into βIII-tubulin+ neurons after induction (Figure 7D), suggesting that other mechanisms are gradually taking over to block reprogramming.


Transcriptional Mechanisms of Proneural Factors and REST in Regulating Neuronal Reprogramming of Astrocytes.

Masserdotti G, Gillotin S, Sutor B, Drechsel D, Irmler M, Jørgensen HF, Sass S, Theis FJ, Beckers J, Berninger B, Guillemot F, Götz M - Cell Stem Cell (2015)

Deletion of REST Removes Reprogramming Block in Astrocytes(A) Schematic representation of the experimental procedure.(B–E) Micrographs of Neurog2ERT2-infected astrocytes (red) with early (B and C) or late (D and E) deletion of REST by infection with a Cre containing viral vector (green) immunostained for the neuronal marker βIII-tubulin (white) at 8 DPI. Yellow arrowheads indicate triple positive cells (DsRed, YFP, βIII-tubulin) while white arrowheads indicate double positive cells (DsRed, GFP). Scale bars, 150 μm.(F) Histogram depicting the proportion of co-transduced double positive cells (red and green) for the astrocytic marker (GFAP, white bars) or the neuronal marker (βIII-tubulin, black bars). Mean ± SEM, three independent biological samples; two-tailed unpaired t test, ∗p < 0.05; ∗∗∗p < 0.001.(G) Postnatal (day 6–7) mouse cortical astrocytes transduced with Ascl1 or Neurog2 are reprogrammed into neurons. However, when cells are maintained longer in culture, increasing levels of H4K20me3 modify the local chromatin environment that becomes favorable to the repressive complex REST. Consequently, Neurog2 fails to access the NeuroD4 promoter. This is bypassed by common downstream transcription factors to both Ascl1 and Neurog2 that are able to generate neurons also in prolonged astrocytic cultures. Unidentified REST co-factors might be recruited to the locus to further remodel the chromatin over time.See also Figure S7.
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Related In: Results  -  Collection

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fig7: Deletion of REST Removes Reprogramming Block in Astrocytes(A) Schematic representation of the experimental procedure.(B–E) Micrographs of Neurog2ERT2-infected astrocytes (red) with early (B and C) or late (D and E) deletion of REST by infection with a Cre containing viral vector (green) immunostained for the neuronal marker βIII-tubulin (white) at 8 DPI. Yellow arrowheads indicate triple positive cells (DsRed, YFP, βIII-tubulin) while white arrowheads indicate double positive cells (DsRed, GFP). Scale bars, 150 μm.(F) Histogram depicting the proportion of co-transduced double positive cells (red and green) for the astrocytic marker (GFAP, white bars) or the neuronal marker (βIII-tubulin, black bars). Mean ± SEM, three independent biological samples; two-tailed unpaired t test, ∗p < 0.05; ∗∗∗p < 0.001.(G) Postnatal (day 6–7) mouse cortical astrocytes transduced with Ascl1 or Neurog2 are reprogrammed into neurons. However, when cells are maintained longer in culture, increasing levels of H4K20me3 modify the local chromatin environment that becomes favorable to the repressive complex REST. Consequently, Neurog2 fails to access the NeuroD4 promoter. This is bypassed by common downstream transcription factors to both Ascl1 and Neurog2 that are able to generate neurons also in prolonged astrocytic cultures. Unidentified REST co-factors might be recruited to the locus to further remodel the chromatin over time.See also Figure S7.
Mentions: To examine the effect of REST deletion on Neurog2ERT2-dependent neuronal conversion, RESTflox astrocytes were co-infected with Neurog2ERT2- and Cre-encoding viruses soon after being plated, or with a 5 day delay (Figure 7A). Cultures were then treated for 3 consecutive days with OHT and analyzed 8 DPI (Figures 7B–7E, Figures S7A–S7D). As previously reported (Xue et al., 2013), REST deletion generated a fraction of βIII-tubulin+ cells on its own without Neurog2ERT2 activation (around 20%, Figure 7F); strikingly, however, 90% of Cre/Neurog2ERT2 transduced cells were βIII-tubulin+ after early REST deletion and delayed Neurog2ERT2 activation (Figure 7D). Delayed Cre-mediated REST deletion still allowed 50% of Cre/Neurog2ERT2 double positive cells to convert into βIII-tubulin+ neurons after induction (Figure 7D), suggesting that other mechanisms are gradually taking over to block reprogramming.

Bottom Line: We found that Neurog2 and Ascl1 rapidly elicited distinct neurogenic programs with only a small subset of shared target genes.Notably, in astrocytes refractory to Neurog2 activation, the underlying neurogenic program remained amenable to reprogramming by exogenous NeuroD4.Our findings support a model of temporal hierarchy for cell fate change during neuronal reprogramming.

View Article: PubMed Central - PubMed

Affiliation: Physiological Genomics, Biomedical Center, University of Munich, 80336 Munich, Germany; Institute for Stem Cell Research, Helmholtz Centre Munich, 85764 Neuherberg, Germany.

No MeSH data available.


Related in: MedlinePlus