Fig. 9

ATF3, KLF7 and ATF3/KLF7/MEF2, but not KLF7/MEF2 induced apoptosis-related genes and specifically caspases. (A) Gene set enrichment analysis (GSEA) was carried out on genes upregulated by each TF combination. Shown is a combined plot of the top 10 enriched gene ontology (biological process) classes for ATF3, KLF7, KLF7/MEF2 and ATF3/KLF7/MEF2, showing normalized enrichment scores (NES) and False Discovery Rate (FDR). The top 10 classes were highly similar between these four groups, and consist mainly of classes relating to immune responses and inflammation. (B) GO classes specifically over-represented (by GSEA) in genes upregulated by KLF7/MEF2 and not ATF3, KLF7 or ATF3/KLF7/MEF2, and vice versa. The latter three groups induced several classes related to caspase activity and apoptosis. Of note all three of these groups induce the class GO:0043065, positive regulation of apoptosis. (C) GSEA enrichment plots for GO:0043065, positive regulation of apoptosis. (D) Expression of the pooled leading edge genes for this GO class in the four TF groups excluding MEF2. Expression of this class is significantly lower in the KLF7/MEF2 group than the other three groups (linear mixed model, with Dunnett’s post-hoc test). (E) Expression of the caspases Casp1, Casp2, Casp5 and Casp9 (the five caspases in the pooled leading edge gene set shown in D) are also lower in KLF7/ MEF2 group (linear mixed model, with Dunnett’s post-hoc test). Yellow dots each represent one caspase, diamonds and grey bars show mean and SEM. F-H. Analysis of predicted targets for ATF3, KLF7 and MEF2 and correlation of log fold-changes (LFC) for each predicted target set with axotomy-induced LFC. Left panels show scatter plots of LFC induced by TFs vs. LFC induced by sciatic nerve injury (SNI) and lines of best fit, while the right panels show and compare the slopes of the lines of best-fit. Of note genes with predicted AP1 sites in their promoters (putative ATF3 targets) were not significantly induced by KLF7/MEF2, in contrast to ATF3, KLF7 and ATF3/KLF7/MEF2. Key: A,B: † FDR < 0.1; * FDR < 0.05. D-H: *p < 0.05, **p < 0.01, ***p < 0.001, (D, E: linear mixed model, Dunnett’s post-hoc tests with comparison to KLF7/MEF2; F-H: linear mixed model; test for slope being not equal to 0; n = 4 animals per group except MEF2 for which n = 3)