Extended Data Figure 2: ACSS2 regulates neuronal gene expression. | Nature

Extended Data Figure 2: ACSS2 regulates neuronal gene expression.

From: Acetyl-CoA synthetase regulates histone acetylation and hippocampal memory

Extended Data Figure 2

a, b, Correlation plots of replicate RNA-seq in undifferentiated CAD cells (a) and differentiated CAD neurons (b) for scramble control. c, Transcriptome analysis via RNA-seq, done in two highly correlated biological replicates, identified 894 genes that became upregulated in differentiated CAD neurons (red dots depict genes with >1.6-fold increase). d, Pathway analysis of the 894 upregulated genes (red dots in Fig. 2a) using StringDB. The protein–protein interaction graph depicts a network of binding partners that centres on key players in activity-dependent signalling and synaptic plasticity: Itpr1, Grin1, Nefh, Dync1h1 and Calm1. e, Gene ontology enrichment analysis shows upregulation of neuronal pathways. Gene ontology analysis was used on the 894 genes that become upregulated in differentiated CAD neurons (Extended Data Fig. 2c; identified by RNA-seq, fold-enrichment (FE) > 3.5, FDR < 0.005). f, Genome browser view of Nudt from RNA-seq and ChIP–seq (H4K12ac, H4K5ac, and H3K9ac: mm10 chr5: 140,327,500–140,339,000). g, Relative gene enrichment of H3K9ac, H4K5ac, and H4K12ac at genes that are upregulated during CAD neuron differentiation (>1.6-fold, grey bars) versus all other genes (black bars). h, i, Correlation plots of replicate RNA-seq in undifferentiated CAD cells for ACL knockdown (h), and ACSS2 knockdown (i). j, k, Correlation plots of replicate RNA-seq in differentiated CAD neurons for ACL knockdown (j) and ACSS2 knockdown (k). l, ACL knockdown has a much smaller effect on differentiation-linked upregulation of neuronal gene expression (compare to Fig. 1d). Scatter plot contrasts the fold-change FPKM of induced genes (Extended Data Fig. 2c) between wild-type and ACL knockdown cells. Marginal distributions show histogram and kernel density estimation. Ordinary least squares linear regression is displayed with 95% confidence interval. m, The corresponding quintiles of upregulated genes (red dots in Extended Data Fig. 2c) with the greatest fold-change FPKM increase in wild-type cells. The ACL knockdown showed the same upward trend as the wild-type cells (red bars, compared to black bars in Fig. 1f), contrasting with the severe defect in ACSS2-knockdown cells (green bars; for each quintile, columns represent the mean induction value of genes and error bars represent s.e.m.). n, Box plot of global mRNA transcript levels in undifferentiated and differentiated CAD neurons from RNA-seq in wild-type (scramble control knockdown; grey), ACSS2-knockdown (shACSS2 #25 knockdown; green), and ACL-knockdown (shACL #17 knockdown; red) cells. Genome-wide transcript levels are reduced in differentiated ACL-knockdown cells when compared to differentiated wild-type cells (error bars, s.e.m.), whereas the global reduction in differentiated ACSS2-knockdown cells is less significant when compared to differentiated wild-type cells (error bars, s.d.). o, Genes sensitive to ACSS2 knockdown (top 20%) are also sensitive to ACSS2i treatment, which lowers their expression compared to all genes.

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