Identification of activity-induced Egr3-dependent genes reveals genes associated with DNA damage response and schizophrenia

Abstract

Bioinformatics and network studies have identified the immediate early gene transcription factor early growth response 3 (EGR3) as a master regulator of genes differentially expressed in the brains of patients with neuropsychiatric illnesses ranging from schizophrenia and bipolar disorder to Alzheimer's disease. However, few studies have identified and validated Egr3-dependent genes in the mammalian brain. We have previously shown that Egr3 is required for stress-responsive behavior, memory, and hippocampal long-term depression in mice. To identify Egr3-dependent genes that may regulate these processes, we conducted an expression microarray on hippocampi from wildtype (WT) and Egr3-/- mice following electroconvulsive seizure (ECS), a stimulus that induces maximal expression of immediate early genes including Egr3. We identified 69 genes that were differentially expressed between WT and Egr3-/- mice one hour following ECS. Bioinformatic analyses showed that many of these are altered in, or associated with, schizophrenia, including Mef2c and Calb2. Enrichr pathway analysis revealed the GADD45 (growth arrest and DNA-damage-inducible) family (Gadd45b, Gadd45g) as a leading group of differentially expressed genes. Together with differentially expressed genes in the AP-1 transcription factor family genes (Fos, Fosb), and the centromere organization protein Cenpa, these results revealed that Egr3 is required for activity-dependent expression of genes involved in the DNA damage response. Our findings show that EGR3 is critical for the expression of genes that are mis-expressed in schizophrenia and reveal a novel requirement for EGR3 in the expression of genes involved in activity-induced DNA damage response.

Fig. 1. EGR3-dependent genes show altered expression in schizophrenia studies.

A Expression heatmap of genes differentially expressed in WT versus Egr3−/− mice following ECS. Expression microarray revealed 69 genes that were differentially expressed in the hippocampus of Egr3−/− mice compared to WT one hour following ECS. The average expression level for each of these genes is shown along a horizontal row in WT (left) and Egr3−/− mice (right) at baseline, and 1 h and 2 h following ECS. (n = 4 animals per condition). B Thirteen published gene expression studies in schizophrenia were queried for the 69 genes that are differentially expressed in Egr3−/− compared with WT mouse hippocampus 1 h following ECS. The heatmap shows normalized log2 fold change values (schizophrenia vs. control) from the 13 published study datasets (vertical columns) for each of the 69 EGR3-dependent genes (horizontal rows).

Fig. 2. Top canonical pathways and biological function categories associated with DEGs generated by the Enrichr web tool.

Enrichr pathway analyses performed on microarray results reveals biological processes associated with the genes that are differentially expressed in hippocampus of Egr3−/− mice compared to WT mice 1 h following ECS. The top 10 enriched pathways from each queried gene set library (A KEGG 2019 Mouse, B Gene Ontology (GO) Biological Process 2021, C WikiPathways 2019 Mouse, D BioPlanet 2019, and E GO Molecular Function 2021) are shown in order from most significant to least significant adjusted p value. The longer and lighter colored the bar, the more significant the pathway term. All pathways presented have significant adjusted p values < 0.05. One of the most significantly enriched pathways in the DEG list was MAPK signaling, specifically including GADD45B and GADD45G. Complete Enrichr results can be found in Supplementary Table S12.

Fig. 3. GADD45 family genes are differentially expressed in Egr3−/− mice.

A, B Expression microarray results of GADD45B pathway gene expression in hippocampus from WT and Egr3−/− mice at baseline (No ECS) and 1 h after ECS. A Gadd45b, B Gadd45g. Quantitative RT-PCR validation of Gadd45b in (C) the original male cohort and (D) the replication female cohort. E qRT-PCR validation of Gadd45g results in original male cohort. (For experiments in A–C and E n = 4 animals/group, experiments in D WT: No ECS, n = 4; 1 h after ECS, n = 5; Egr3−/−: No ECS, n = 4; 1 h after ECS, n = 5; *p < 0.05, **p < 0.01, ***p < 0.001, controlled for multiple comparisons, values represent means ± SEM). Statistical analyses for these, and all subsequent graphs, are shown in Table 3.

Fig. 4. Numerous genes are differentially expressed in Egr3−/− mice compared with WT mice following ECS.

Microarray analysis results (i, n = 4 animals/group) and follow-up qRT-PCR results performed on the original RNA samples used in the microarray (ii, n = 4 animals/group) and in a replication cohort of female mice (iii, WT: No ECS, n = 4; 1 h after ECS, n = 5; Egr3−/−: No ECS, n = 4; 1 h after ECS, n = 5). A Cenpa, the most highly DEG in the dataset, AP-1 family genes B Fos and C Fosb, and additional memory-related gene D Nr4a2, which is also involved in DNA repair. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, controlled for multiple comparisons, values represent means ± SEM).

Fig. 5. Mef2c and Calb2 display unique patterns of regulation.

Microarray analysis results (A, C) and follow-up qRT-PCR results in males (B, D) for schizophrenia associated gene Mef2c (A, B) and Calb2 (C, D), genes overexpressed in schizophrenia. (n = 4 animals/group; ANOVAs were significant for each (see Table 3), p-values represent post-hoc analyses, **p < 0.01, ***p < 0.001, controlled for multiple comparisons, values represent means ± SEM).