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

Figure 6.

Figure 6

Chromatin Marks and REST Binding at Regulatory Regions of the Downstream Targets NeuroD4, NeuroD1, and Sox11

(A and A′) H4K20me3 μChIP-PCR on immunoprecipitated material from astroglia cultures collected 1 day or 6 days after being plated as indicated in the scheme at top of (A).

(B and B′) Analysis of REST binding to NeuroD4 by μChIP-PCR on immunoprecipitated samples from short-term astroglia cultures as indicated in the scheme in (B). Amplification of the REST binding element within the NeuroD1 intron was used as a positive control while a region within the promoter of Sox11 was used as a negative control. Percentages of input chromatin were quantified in duplicate from three independent biological samples (mean ± SEM).

(C and C′) REST μChIP-PCR on immunoprecipitated samples from Neurog2ERT2-transduced astrocytes cultured for shorter or longer periods and treated with OHT for 24 hr as indicated at the top of (C). REST ChIP values were normalized to their respective mock ChIP values (mean ± SEM in duplicate from three independent biological samples; two-tailed unpaired t test, ∗p < 0.05).

(D and D′) HA-Neurog2ERT2 μChIP-PCR on immunoprecipitated genomic DNA from delayed astroglia cultures. RESTflox cKO were transduced with Neurog2ERT2 and adeno-Cre virus with a late OHT induction as indicated (D). The Atoh8 promoter and NeuroD1 promoter regions were used as controls for the effect of REST deletion on Neurog2 binding. Percentages of input chromatin were quantified in duplicate from three independent biological samples (mean ± SEM; two-tailed unpaired t test, ∗p < 0.05).

(E and E′) Real-time qPCR analysis on Neurog2ERT2-astrocytes treated with OHT for 48 hr after early or late REST Cre-mediated deletion as indicated at the top of the histogram (E). Control samples (Cre- OHT+) were transduced with adeno null virus 1 day after being seeded at the same time as the delayed Cre sample (adeno-Cre virus, Early Cre+OHT+). In parallel, another set of cells was transduced with adeno-Cre virus 5 days later (Delayed Cre+OHT+). Mean ± SEM in duplicate from three independent culture batches.

See also Figure S6.