Broad domains of histone 3 lysine 4 trimethylation are associated with transcriptional activation in CA1 neurons of the hippocampus during memory formation

Abstract

Transcriptional changes in the hippocampus are required for memory formation, and these changes are regulated by numerous post-translational modifications of chromatin-associated proteins. One of the epigenetic marks that has been implicated in memory formation is histone 3 lysine 4 trimethylation (H3K4me3), and this modification is found at the promoters of actively transcribed genes. The total levels of H3K4me3 are increased in the CA1 region of the hippocampus during memory formation, and genetic perturbation of the K4 methyltransferases and demethylases interferes with forming memories. Previous chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) analyses failed to detect changes in H3K4me3 levels at the promoters of memory-linked genes. Since the breadth of H3K4me3 marks was recently reported to be associated with the transcriptional outcome of a gene, we re-analyzed H3K4me3 ChIP-seq data sets to identify the role of H3K4me3 broad domains in CA1 neurons, as well as identify differences in breadth that occur during contextual fear conditioning. We found that, under baseline conditions, broad H3K4me3 peaks mark important learning and memory genes and are often regulated by super-enhancers. The peaks at many learning-associated genes become broader during novel environment exposure and memory formation. Furthermore, the important learning- and memory-associated lysine methyltransferases, Kmt2a and Kmt2b, are involved in maintaining H3K4me3 peak width. Our findings highlight the importance of analyzing H3K4me3 peak shape, and demonstrate that breadth of H3K4me3 marks in neurons of the hippocampus is regulated during memory formation.

Keywords: Bioinformatics; Epigenetics; Hippocampus; Histone methylation; Memory; Transcription.

Figure 1. H3K4me3 broad domains in neurons of the hippocampus mark promoters of genes that are cell type-specific and highly expressed.

A) Data collection strategy for source data. CA1 region of hippocampus from cKO mice or mice that went through CFC was dissected, nuclei were isolated, and neuronal nuclei were sorted using NeuN antibody (Halder et al., 2016; Kerimoglu et al., 2017). Publicly available data from these studies were downloaded, and we conducted breadth analysis of H3K4me3 peaks and assigned regions of enrichment to annotated promoters. B) Scatter plot of H3K4me3 breadth in kilobases (kb) and H3K4me3 intensity for naïve WT mice. Genes uniquely marked by top 5% broadest H3K4me3 peaks are marked in blue, genes uniquely marked by H3K4me3 peaks with top 5% intensity are red, and genes fitting both criteria are in purple. Genes fulfilling neither criterion are marked in black. C) Heatmaps of H3K4me3 across all genes, broad genes, and intense genes. Heatmaps are centered at gene TSS and span +/− 5 kb. Yellow indicates higher H3K4me3 levels. D) Boxplot of RNA expression levels of control gene set, broad genes, and intense genes. Control peaks were a set of genes of the same number as the broad and sharp gene sets that were randomly selected from genes that were neither broad nor sharp but had H3K4me3 at their promoters. Horizontal lines in the middle of boxes indicate median expression level. * = p < 0.05, *** = p < 1e-8 by Wilcoxon rank sum with Bonferroni correction. E) DAVID GO analysis of broad genes. F) DAVID GO analysis of intense genes. Biological process (BP) terms for genes with p < 0.05. Bonferroni-corrected p value shown.

Figure 2. Genes regulated by super-enhancers harbor H3K4me3 broad domains in hippocampal neurons.

A) Boxplot of RNA expression levels for a control gene set, E-reg genes, and SE-reg genes. Control peaks were a set of genes of the same number as the E-reg gene set that was randomly selected from genes that were not part of the E-reg or SE-reg gene sets. B) Density plot showing the distribution of H3K4me3 breadth values for control genes, E-reg genes, and SE-reg genes. * = p < 0.05, ** = p < 0.01, *** = p < 1e-10 by Wilcoxon rank sum with Bonferroni correction.

Figure 3. H3K4me3 breadth is dynamically regulated at gene promoters in CA1 neurons by CFC.

A) Tracks of representative genes increasing in H3K4me3 intensity (top) and breadth (bottom) with context and context plus shock relative to naïve. Scale is normalized across conditions by sequencing library size, and data was binned into 20 base pair windows. Horizontal black line marked 1 kb gives scale for all gene tracks. B) Metagene profiles averaging signal of H3K4me3 across all significantly activated genes relative to naïve from RNA-seq in context (top) or context + shock (bottom). C) Metagene profiles of H3K4me3 signal in naïve mice at genes that become activated in context or context plus shock relative to a control group of genes that was randomly selected from genes with no significant expression change in RNA-seq.

Figure 4. Genes exhibiting increased H3K4me3 breadth with CFC are learning-related and highly induced.

A) Boxplots of expression changes relative to naïve for broad and broadening genes, sharp and more intense genes, and a control gene set of the same size. B) GO analysis of broad and broadening and sharp and more intense gene sets for context (top) and context plus shock (bottom) conditions. Biological process (BP) terms meeting significance level of Bonferroni-corrected p < 0.05 are shown. Heatmaps reflect −log10 of Bonferroni-corrected p-value. C) Bar plots of agreement between broad and broadening genes with context or context plus shock relative to naïve. If context or context plus shock peak breadth was less than 25 base pairs different up or downstream of the naïve peak boundary, it was classified as not different. ** = p < 1e-9, *** = p < 1e-15 by Wilcoxon rank sum with Bonferroni correction.

Figure 5. H3K4 methyltransferases Kmt2a and Kmt2b globally regulate H3K4me3 breadth in hippocampal neurons.

A) Density plots of H3K4me3 breadth for all genes in control, Kmt2a cKO, and Kmt2b cKO mice. B) Genes with significant narrowing of H3K4me3 breadth in Kmt2a cKO (top right panel) or Kmt2b cKO (bottom right panel) relative to control mice (left panels). C) Density plots of H3K4me3 intensity in control, Kmt2a cKO, and Kmt2b cKO mice. D) Breadth of peaks in control mice that narrow after cKO of Kmt2a or Kmt2b. E) Overlap of genes that narrow after cKO of Kmt2a or Kmt2b and genes that broaden in context or context plus shock relative to naïve. *** = p < 1e-15 by Wilcoxon rank sum.