Loss of NSD2 causes dysregulation of synaptic genes and altered H3K36 dimethylation in mice

Abstract

Background: Epigenetic disruptions have been implicated in neurodevelopmental disorders. NSD2 is associated with developmental delay/intellectual disability; however, its role in brain development and function remains unclear. Methods: We performed transcriptomic and epigenetic analyses using Nsd2 knockout mice to better understand the role of NSD2 in the brain. Results and discussion: Transcriptomic analysis revealed that the loss of NSD2 caused dysregulation of genes related to synaptic transmission and formation. By analyzing changes in H3 lysine 36 dimethylation (H3K36me2), NSD2-mediated H3K36me2 mainly marked quiescent state regions and the redistribution of H3K36me2 occurred at transcribed genes and enhancers. By integrating transcriptomic and epigenetic data, we observed that H3K36me2 changes in a subset of dysregulated genes related to synaptic transmission and formation. These results suggest that NSD2 is involved in the regulation of genes important for neural function through H3K36me2. Our findings provide insights into the role of NSD2 and improve our understanding of epigenetic regulation in the brain.

Keywords: ChIP sequencing; H3K36me2; NSD2; RNA sequencing; neurodevelopmental disorder.

FIGURE 1

Loss of NSD2 affects gene expression in the brain. (A) Volcano plot of RNA sequencing analysis showing differentially expressed genes (DEGs) between Nsd2 knockout (KO) and wild-type (WT) male mouse brains at E15.5; n = 3 in each group. DEGs are labeled in blue (downregulated) and red (upregulated). The horizontal line indicates the false discovery rate (FDR) threshold of 0.05. (B, C) Bar plots showing enriched Gene Ontology terms among biological processes related to downregulated DEGs (B) and upregulated DEGs (C) in Nsd2 KO males (p < 0.05). (D) Heatmap showing clustered protocadherin genes differentially expressed in Nsd2 KO males by hierarchical clustering of WT and Nsd2 KO biological replicates. (E) Venn diagram showing the overlap between human ortholog DEGs in Nsd2 KO male and neurodevelopmental disorder gene sets, including autism spectrum disorder from SFARI (left), intellectual disability (middle), and schizophrenia (right). ASD, autism spectrum disorder; ID, intellectual disability.

FIGURE 2

Genome-wide changes in H3K36me2. (A) Scatterplot showing normalized H3K36me2 chromatin immunoprecipitation (ChIP) signal for the 10 kb window in wild-type (WT) and Nsd2 knockout (KO) E15.5 brains. The windows with loss or gain of H3K36me2 were defined as normalized H3K36me2 ChIP signal ratio between Nsd2 KO versus WT E15.5 brain <1/1.5 or >1.5 and labeled in blue (loss) and red (gain). (B) Genome browser snapshot showing representative H3K36me2 ChIP signal in WT and Nsd2 KO E15.5 brains. H3K36me2 loss/gain in Nsd2 KO are shown as red and blue bars, respectively, and genes (from RefSeq) are annotated at the bottom. (C) Bar plot showing the ratio of observed-to-random H3K36me2 loss and gain in annotated genomic regions in Nsd2 KO E15.5 brains. (D) Bar plots showing enrichment analysis of ChromHMM annotation for H3K36me2 loss (left) and gain (right) in Nsd2 KO E15.5 brains. Asterisks indicate significance (q value <0.05) for background versus H3K36me2 loss/gain using Fisher’s exact test.

FIGURE 3

Relationship between gene expression and H3K36me2 levels. (A) Plot showing averaged H3K36me2 chromatin immunoprecipitation (ChIP) signal grouped into quartiles (Q4-Q1) and zero by transcripts per million and zero value in wild-type (WT) and knockout (KO) E15.5 brains. (B) Heatmaps showing H3K36me2 ChIP signal enrichment of differentially expressed genes (DEGs). (C) Genome browser snapshot showing representative H3K36me2 ChIP signal in WT and Nsd2 knockout (KO) E15.5 brains. The shaded areas indicate Lrfn5 (downregulated DEG) and (D) Pcdhb genes and Sorbs2 (upregulated DEGs). H3K36me2 loss/gain in Nsd2 KO are shown as red and blue bars, respectively, and genes (from RefSeq) are annotated at the bottom. TSS, transcription start site; TES, transcription end site.