Extended Data Fig. 3: AraC induces expression of immediate early genes and late response genes in cortical neurons.
a, Genome browser screenshot showing a double-strand break (END-seq) induced by AraC (40 μM) that colocalizes with the enhancer mark H3K4me1 (non-treated) in primary cortical neurons. b, Heatmaps of END-seq (DSB) in non-treated and AraC-treated primary cortical neurons, and ChIP-seq of H3K4me1 in non-treated primary cortical neurons. The peaks are ordered by END-seq intensity and plotted 1 kb on either side of DSB summits. c, Composite DNA sequence motif analysis 4 bp on either side of AraC-END-seq peak summits in primary cortical neurons. Peak fraction and p value are indicated. Statistical significance was determined using the two-sided binomal test. d, Motif analysis by HOMER. Top, the Fos binding motif downloaded from JASPAR motif database. Bottom, the most enriched motif identified from sequences within 200 bp of the AraC-END-seq peak summits in primary cortical neurons. Peak fraction and p value are indicated. Statistical significance was determined using the two-sided binomal test. e, Aggregate plot showing the Fos motif distribution at AraC-END-seq peak summits in primary cortical neurons 1 kb on either side of AraC-END-seq peak summits. f, Volcano plot showing differentially expressed genes upon AraC treatment in primary cortical neurons. Genes whose expression were upregulated and downregulated significantly (log2fold change>1 and p value < 0.01) are indicated in red and blue respectively. The 10 most significantly upregulated genes are indicated. Statistical significance was determined using the two-sided Wald test and adjusted by the Benjamini & Hochberg method. g, Heatmap of immediate early genes (IEGs) and late response genes (LRGs) expression in non-treated and AraC-treated primary cortical neurons. h, Top 15 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways identified from upregulated genes upon AraC treatment in primary cortical neurons. Statistical significance was determined using the one-sided hypergeometric test. i, qRT-PCR measurement of Fos, Fosb, Npas4, Junb, Bdnf and Vgf gene expression in non-treated or AraC/dFdC (40 μM for 16 hr) treated primary cortical neurons. Statistical significance was determined using the two-sided paired t test, p value is indicated. Data are presented as mean ± SD, n = 3 biologically independent samples. j, qRT-PCR measurement of Fos and Npas4 gene expression in non-treated or etoposide (ETO, 50 μM for 16 hr) treated primary cortical neurons. Statistical significance was determined using two-sided paired t test, p value is indicated. Data are presented as mean ± SD, n = 3 biologically independent samples. k, qRT-PCR measurement of Fos, Fosb, Npas4, Junb, Bdnf and Vgf gene expression in wild-type or Tdg−/− primary cortical neurons treated with AraC (40 μM for 16 hr). 4-Hydroxytamoxifen (4-OHT) was added in vitro to deplete TDG in primary cortical neurons isolated from CreERT2 Tdgfl/fl embryos. Statistical significance was determined using two-sided paired t test, p value is indicated. Data are presented as mean ± SD, n = 3 biologically independent samples.
