Neuronal Histone Methyltransferase EZH2 Regulates Neuronal Morphogenesis, Synaptic Plasticity, and Cognitive Behavior in Mice

The histone methyltransferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2)-mediated trimethylation of histone H3 lysine 27 (H3K27me3) regulates neural stem cell proliferation and fate specificity through silencing different gene sets in the central nervous system. Here, we explored the function of EZH2 in early post-mitotic neurons by generating a neuron-specific Ezh2 conditional knockout mouse line. The results showed that a lack of neuronal EZH2 led to delayed neuronal migration, more complex dendritic arborization, and increased dendritic spine density. Transcriptome analysis revealed that neuronal EZH2-regulated genes are related to neuronal morphogenesis. In particular, the gene encoding p21-activated kinase 3 (Pak3) was identified as a target gene suppressed by EZH2 and H3K27me3, and expression of the dominant negative Pak3 reversed Ezh2 knockout-induced higher dendritic spine density. Finally, the lack of neuronal EZH2 resulted in impaired memory behaviors in adult mice. Our results demonstrated that neuronal EZH2 acts to control multiple steps of neuronal morphogenesis during development, and has long-lasting effects on cognitive function in adult mice. Supplementary Information The online version contains supplementary material available at 10.1007/s12264-023-01074-1.

and visualized using Metascape (http://metascape.org) and Cytoscape5.The program constructs a GO term network among terms with a similarity >0.3, which are connected by edges.Each node represents one enriched GO term, where node size is the number of genes within a GO term.GO terms within the same cluster are visualized with the same color.P-values of the enrichments refer back to Fig. 6, which shows the maximal P-value is 1.0 × 10 −3 .A GO term network of down-regulated genes in the Nestin-Cre;Ezh2 f/f compared with the control.B GO term network of down-regulated genes in the Neurod6 (Nex)-Cre;Ezh2 f/f compared with the control.C GO term network of up-regulated genes in the Nestin-Cre; Ezh2 f/f compared with the control.D GO term network of up-regulated genes in the Neurod6 (Nex)-Cre;Ezh2 f/f compared with the control.A Quantification of time spent in the open arms of the elevated plus maze (female: n = 10 mice for control mice and n = 16 for Ezh2 Δ/Δ mice, P = 0.6808; male: n = 15 for control mice and n = 9 for Ezh2 Δ/Δ mice, P = 0.6093, unpaired Student's t-test).B Quantification of time spent in the closed arms of the elevated plus maze (female: n = 10 for the control group and n = 16 for the Ezh2 Δ/Δ group, P = 0.6808; male: n = 15 for control mice and n = 9 for Ezh2 Δ/Δ mice, P = 0.6093, unpaired Student's ttest).C-E Quantification of alterations in time spent in the peripheral zone (C), central zone (D), and rearing behavior (E).Female: n = 10 for control mice and n = 16 for Ezh2 Δ/Δ mice; male: n = 15 for control mice and n = 9 for Ezh2 Δ/Δ mice.Open field peripheral: P = 0.8551 for female mice and P = 0.2052 for male mice, unpaired Student's t-test.Open field center: P = 0.8561 for female mice and P = 0.3643 for male mice, unpaired Student's t-test.Open field rearing: P = 0.4703 for female mice and P = 0.1854 for male mice, unpaired Student's t-test.Data are presented as the mean ± SEM. n.s., no significant difference, compared to control if not designated.

Fig. S1
Fig. S1 Expression of EZH2, SUZ12, and H3K27me3 in cultured excitatory neurons.A Representative images showing the expression of EZH2, SUZ1, and H3K27me3 in cultured primary cortical neurons.Yellow arrowheads indicate Tuj1-positive neurons.Scale bar, 50 μm.B Upper: representative images of a P0 mouse coronal brain section stained with anti-Cre antibody showing the expression of Cre in the cortical and hippocampal regions.Lower: enlarged images of the two white dashed boxes in the upper panel.Scale bar, 500 µm (lower) and 2 mm (upper).C NeuroD6-Cre mediated recombination in the adult mouse brain is confirmed by RT-PCR.Representative PCR image showing a 254-bp fragment band generated by NeuroD6-Cre-mediated Ezh2 deletion in Neurod6-Cre/Ezh2 f/+ heterozygous mice.The 587-bp band is the remaining wild-type EZH2 band.D Representative Western blot image (left) and quantification (right) showing the reduction of EZH2 by measuring the ratio of EZH2 and H3.The data are normalized to the control (P = 0.0117, n = 5 mice for each condition, unpaired Student's t-test).Data are represented as the mean ± SEM. *P <0.05, compared to control if not designated.

Fig. S2
Fig. S2 Deletion of EZH2 manifests no structural brain abnormalities.A Representative Nissl staining

Fig. S3
Fig. S3 Loss of EZH2 in cortical neurons delays the migration of upper cortical neurons.A Representative images of cortex immunostained for Foxp1 and Ctip2 in control and Ezh2 ∆/∆ mice at P0. Foxp1 and Ctip2 labeling help to define layers II-IV and IV-V of the cortical plate, respectively.The distributions of labeled cells are assigned into 6 bins across the apicobasal axis.Scale bar, 100 μm.B Quantification of Foxp1-positive cells in 6 equal bins showing significantly reduced Foxp1-

Fig. S4
Fig. S4 Lack of EZH2 has little effect on cortical neuronal migration at P7. Representative confocal images of mouse cortices in utero electroporated with dsRED or dsRED/Neurod1-Cre.The electroporation was performed at E15 and the pups were harvested at P7. Scale bar, 200 μm.

Fig. S5
Fig. S5 Network of enriched GO terms among each differentially-expressed gene set depicts connections among GO terms.Subgroups of the enriched GO terms shown in Fig. 6 have been plotted

Fig
Fig. S6 Ezh2 Δ/Δ mutant mice show little defects in the elevated plus maze test and the open field test.