Fig. 8: Locus-specific acetylation restores NeuroD2 transcription.

a Snap view of the Neurod2 locus. The profiles for CBP and p300 binding, H3K27 acetylation and ATAC-seq signal are shown. The bottom orange track corresponds to the NeuroD2 ChIP-seq data generated in ref. ²⁹. A scheme presenting the strategy used to drive the KAT activity of p300 to the Neurod2 promoter using a fusion protein with dCas9 is also shown. The positions targeted by the gRNAs (red lines, gND2 A-D) are indicated. Note that the target regions are in the proximity of bHLH sites. b Scheme of the co-infection of LVs expressing cre recombinase, dCas9-KAT, and the Neurod2 gRNAs (A-D mix). c Representative image of NeuroD2 protein levels in the cells co-infected with LV-CRE-GFP and the lentiviruses LV-gRNA-mCherry specific for Neurod2 (white arrows). As a specificity control, we conducted the same experiment using a gRNA targeted to Hpca (blue arrows) (n = 4 wells per condition in 2 PNCs). As a comparison, cells infected with LV-CRE-GFP alone (yellow arrows) show strongly diminished NeuroD2 levels as well. Scale bar: 50 μm. d Quantification of different cell subpopulations observed in the experiment shown in panel (c) (n = 4 wells per condition in 2 PNCs). Two-tailed t-test: ****p < 0.0001, ***p < 0.001, **p < 0.01; *p < 0.05. Data are presented as mean values ± SEM. e Scheme of rescue experiment with plasmids carrying dCas9-KAT and the Neurod2 gRNA in dKAT3-KO cells that had already lost Neurod2 expression. f Representative image of NeuroD2 expression in control (PNC + LV-GFP) and dKAT3-depleted (PNC + LV-CRE-GFP) neurons after transfection with dCas9-KAT and gND2-C targeting the NeuroD2 promoter. Arrows indicate LV-CRE-GFP infected cells transfected with the gRNA-carrying vector. A gRNA targeting the hippocalcin promoter was used as a specificity control. Scale: 20 μm. g Quantification of the percentage of transfected cells showing normal NeuroD2 expression after transfection with gND2-C alone and dCas9-KAT co-transfected with gND2-C, gND2-A, gND2-B, or gRNA control independently (experiments in three independent PNCs; n = 30–40 neurons per condition). Source data for graphs in panels (d) and (g) are provided as a Source data file.