Journal: Nature Communications

Article Title: KAT3-dependent acetylation of cell type-specific genes maintains neuronal identity in the adult mouse brain

doi: 10.1038/s41467-020-16246-0

Figure Lengend Snippet: a Cumulative graph showing the log2 fold-change value of DEGs in dKAT3-ifKOs (mRNA-seq, 1 month after TMX, n = 3 per genotype). Upregulated genes are presented in red and downregulated genes in blue ( p .adj < 0.05 and |log2FC| ≥ 1). The bottom bar graph compares the area in each set. b Volcano plots of RNA-seq analysis. From left to right, we present all genes (left) and the subset of housekeeping genes listed in ref. (right). Gray: genes that are not significantly deregulated; red: upregulated genes; blue: downregulated genes; purple: housekeeping genes. c The 10 most enriched categories identified by Gene Ontology (GO) analysis on up- (red) and downregulated (blue) genes with a p .adj < 0.05 and |log2FC| ≥ 1. The upregulated gene set only retrieved three categories. d RNA-seq profiles for three representative neuronal-specific genes: Rbfox3 (NeuN), Hpca , and Camk4 . Scale: 2 kb. e Immunohistochemistry against the neuronal protein encoded by Camk4 , Rbfox3 (NeuN), and Hpca . Dashed line labels the position of the stratum pyramidale based on DAPI images ( n = 3). Scale: 60 μm. f Loss of NeuN immunoreactivity in DG neurons infected with a cre recombinase-expressing AAV (GFP + ). The experiment was performed 3 times with different sets of mice. The virus was injected unilaterally into the DG of adult Crebbp f/f ::Ep300 f/f mice and the mice were perfused 1 month later (Supplementary Fig. ). See Supplementary Fig. for immunostaining against CBP in the same brain slide. Scale: 200 μm. g Scheme representing the strategy to eliminate both KAT3 proteins in hippocampal PNCs from E17 dKAT3 f/f embryos. h Representative images showing morphological changes in Crebbp f/f ::Ep300 f/f hippocampal neurons infected with LV-CRE compared with LV-GFP control ( n = 6 in both groups). i RT-PCR demonstrates decreased levels of Neurod2 (ND2), Hpca (hippocalcin), and Rbfox3 (NeuN) transcripts in dKAT3-KO PNCs. In contrast, several housekeeping genes ( Ppl1 , Pgk1 , Rpl23 ) are unaffected ( n = 5–6 in both groups). Data are presented as mean values ± SEM. Two-tailed t -test: **** p < 0.0001, *** p < 0.001, ** p < 0.01. Source data for graphs in panel ( i ) are provided as a file.

Article Snippet: The following lentiviral constructs were acquired from Addgene: LV-CRE-GFP (Addgene #20781), LV-CRE-mCherry (Addgene #27546), and LV-RFP (Addgene #17619) were used to label neurons and astrocytes with fluorescent reporter proteins; dCas9-KAT (Addgene #61357), LV-dCas9-KAT (Addgene #83889), and LV-U6gRNA-Cherry (Addgene #85708) were used in epi-editing experiments; and LV-phND2-N174 (Addgene #31822) was used to overexpress human NeuroD2.

Techniques: RNA Sequencing Assay, Immunohistochemistry, Infection, Expressing, Injection, Immunostaining, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Nature Communications

Article Title: KAT3-dependent acetylation of cell type-specific genes maintains neuronal identity in the adult mouse brain

doi: 10.1038/s41467-020-16246-0

Figure Lengend Snippet: a Hippocampal PNCs from E17 Crebbp f/f :: Ep300 f/f embryos were co-transfected with constructs that drive the expression of the Cre recombinase and full-length CBP. b Plasmid combination to express recombinant CBP simultaneously to endogenous CBP and p300 ablation. c Representative images of NeuroD2 and hippocalcin staining in PNC transfected with the constructs shown in ( b ). Note the reduced expression in the GFP+ cells in the absence of heterologous full-length CBP (experiments in two independent PNCs). Scale: 10 μm. d Quantification of the percentage of NeuroD2-positive or -negative cells and hippocalcin-positive or -negative cells among all GFP-positive cells ( n = 60 neurons per condition). e Scheme of the CBP fragments used for rescuing NeuroD2 expression (see Methods for additional details). NLS: nuclear localization domain; KAT: acetyltransferase domain; KIX: kinase-inducible domain interacting domain; bHLH-i: region of interaction with bHLH transcription factors. f Hippocampal PNCs from E17 Crebbp f/f :: Ep300 f/f embryos were transfected with constructs that drive the expression of the Cre recombinase and the different CBP fragments and the KAT domain shown in panel ( e ). g Representative images of NeuroD2 staining in PNCs infected with LV-CREw/oGFP and transfected with the different domains of CBP shown in panel ( e ). PNCs infected with LV-GFP (i.e. with wild type phenotype) were used as a control for baseline NeuroD2 level (2 independent PNCs). Scale: 20 μm. h Quantification of the percentage of NeuroD2-positive or -negative cells among all GFP-positive neurons ( n = 30–80 neurons per condition). Source data for graphs in panels ( d ) and ( h ) are provided as a file.

Article Snippet: The following lentiviral constructs were acquired from Addgene: LV-CRE-GFP (Addgene #20781), LV-CRE-mCherry (Addgene #27546), and LV-RFP (Addgene #17619) were used to label neurons and astrocytes with fluorescent reporter proteins; dCas9-KAT (Addgene #61357), LV-dCas9-KAT (Addgene #83889), and LV-U6gRNA-Cherry (Addgene #85708) were used in epi-editing experiments; and LV-phND2-N174 (Addgene #31822) was used to overexpress human NeuroD2.

Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Recombinant, Staining, Infection

Journal: Nature Communications

Article Title: KAT3-dependent acetylation of cell type-specific genes maintains neuronal identity in the adult mouse brain

doi: 10.1038/s41467-020-16246-0

Figure Lengend Snippet: 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. . 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 file.

Article Snippet: The following lentiviral constructs were acquired from Addgene: LV-CRE-GFP (Addgene #20781), LV-CRE-mCherry (Addgene #27546), and LV-RFP (Addgene #17619) were used to label neurons and astrocytes with fluorescent reporter proteins; dCas9-KAT (Addgene #61357), LV-dCas9-KAT (Addgene #83889), and LV-U6gRNA-Cherry (Addgene #85708) were used in epi-editing experiments; and LV-phND2-N174 (Addgene #31822) was used to overexpress human NeuroD2.

Techniques: Binding Assay, ChIP-sequencing, Generated, Activity Assay, Infection, Expressing, Two Tailed Test, Transfection, Plasmid Preparation