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. 2015 Jul 2;17(1):74–88. doi: 10.1016/j.stem.2015.05.014

Figure 7.

Figure 7

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.