Fig 1: Verification of NEUROD2 binding to the conserved element within Stim1 intron 2. A, NEUROD2 binding to Stim1 intronic element is confirmed in E14.5 and P0 cortices by ChIP-qPCR. ChIP DNA acquired with an unrelated GFP antibody is used as a negative control. Amount of DNA immunoprecipitated with either a NEUROD2 antibody (NEUROD2 ChIP DNA) or GFP antibody (GFP ChIP DNA) is expressed as percentage of input DNA (% input). NEUROD2 % input values are then normalized to GFP % input values. Strong enrichment of NEUROD2 is detected at the NEUROD2 binding element located in Stim1 intron 2 both in E14.5 and P0 cortices. Slight enrichments are observed for Stim1 introns 1 and 3. Data are representative of six biological replicates each composed of three technical replicates. Bars represent SEM. p < 0.0001 determined by one-way ANOVA followed by unpaired t test, *p < 0.05, **p < 1 × 10-4, ***p < 1 × 10-5 (Table 2). B, Luciferase activity is measured from HEK 293T cell lysates that are transfected either with an empty luciferase reporter plasmid (pXPG) or with a luciferase reporter downstream of a wild-type (WT-570) or mutated 570 bp fragment (MUT-570) Stim1 intronic element. In addition, cells are also cotransfected with either an empty (pc4) or NEUROD2 expressing (ND2) pcDNA4 vector. Firefly luciferase activity is normalized to Renilla luciferase signal. Data represent three independent experiments with each sample measured in triplicates. Bars represent SEM. D’Agostino–Pearson test showed normal distribution of the data (a = 0.05). One-way ANOVA and post hoc Tukey’s multiple-comparison analysis was performed, ****p < 0.0001 (Table 2).
Fig 2: Overexpression of Neurod2 reduces the SOCE response. A, Primary cortical neurons were transfected with either empty or resND2 expressing pcDNA4 vector, and calcium imaging was performed as described in Figure 6A. B, C, Measurement of peak heights of first- and second-wave of Ca2+ signals revealed that overexpression of Neurod2 in otherwise wild-type neurons causes a suppression of SOCE but does not affect steady-state levels of ER Ca2+. D–F, Calculation of total area under peaks demonstrated that both late and early phases of SOCE are downregulated upon Neurod2 overexpression. Traces are color coded as follows: pcDNA4 (blue) and resND2 (orange). Bars represent the SEM. Unpaired t test determined the p value: *p < 0.05. Data are presented as bar graphs; the line marks the median; the box represents the 25th and 75th percentiles; top and bottom whiskers mark minima and maxima, respectively (Table 2). ND2, Neurod2.
Fig 3: Full-length CBP is required to restore neuronal-specific transcription.a Hippocampal PNCs from E17 Crebbpf/f::Ep300f/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 Crebbpf/f::Ep300f/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 Source data file.
Fig 4: RNA-seq analysis of primary cortical neurons silenced for Neurod2 expression. (A) RNA-Seq analysis was carried out on primary cortical neurons electroporated with a NS (non-silencing) shRNA or one of the two independent shRNAs against Neurod2. 25 genes were down-regulated and 9 were up-regulated upon knockdown of Neurod2. fold-change > 1.5, FDR < 0.05. (B) A protein interactome network of REELIN revealed additional genetic targets of NEUROD2 that also interact with REELIN. Interactome analysis was based on the String Database34,35.
Fig 5: NEUROD2 binding to introns along the Reln gene is associated with suppression of Reln gene expression. (A,B) Four prominent intronic NEUROD2 binding sites are plotted on the Reln gene. Ab1, Ab2 and Ab3 represent ChIP experiment carried out with three separate NEUROD2 antibodies. Several different histone modifications corresponding to promoters (H3K4me3), enhancers (H3K4me1), actively transcribed (H3K27ac and H3K36me3) or repressed (H3K9me3 and H3K27me3) chromatin are not enriched in NEUROD2 binding sites. A slight enrichment of CTCF binding within intron 3 of Reln is detected. (C) NEUROD2 binding to all four intronic regions is confirmed by ChIP-qPCR. Immunoprecipitation with an unrelated GFP antibody is used as a negative control. n = 12 (three biological x four technical replicates). Bars represent S.E.M. *p < 0.05, **p < 0.005, ***p < 0.0001 by two-tailed unpaired t-test. (D) RT-qPCR analysis of Reln gene expression in primary cortical neurons reveals significant upregulation upon knockdown of Neurod2 with two separate shRNAs. Gapdh mRNA was used as a normalization control. n = 9 (three biological x three technical replicates). p < 0.0001 by one-way ANOVA. ***p < 0.0001, **p < 0.01 by post hoc Sidak multiple comparison tests. Bars represent S.E.M. (E) Immunoblotting of REELIN from 5 DIV primary cortical cultures electroporated with NS, shNeurod2-1 or with shNeurod2-1 together with a plasmid expressing an shRNA resistant Neurod2 cDNA (resNd2). The protein blot has been probed with REELIN antibody and HSP90 antibody as a loading control. Cropped parts of the gel are indicated with a line. (F) Quantification of REELIN protein amounts from five independent experiments. Data are presented after normalizing to HSP90 loading control. *p = 0.02 by two-tailed unpaired t-test. Abbreviations: NS (non-silencing), shNd2-1 (shNeurod2-1), shNd2-2 (shNeurod2-2), resNd2 (rescue Neurod2), ns (non-significant).
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