Histone bivalency regulates the timing of cerebellar granule cell development

Abstract

Developing neurons undergo a progression of morphological and gene expression changes as they transition from neuronal progenitors to mature, multipolar neurons. Here we use RNA-seq and H3K4me3 and H3K27me3 ChIP-seq to analyze how chromatin modifications control gene expression in a specific type of CNS neuron, the mouse cerebellar granule cell (GC). We find that in proliferating GC progenitors (GCPs), H3K4me3/H3K27me3 bivalency is common at neuronal genes and undergoes dynamic changes that correlate with gene expression during migration and circuit formation. Expressing a fluorescent sensor for bivalent H3K4me3 and H3K27me3 domains revealed subnuclear bivalent foci in proliferating GCPs. Inhibiting H3K27 methyltransferases EZH1 and EZH2 in vitro and in organotypic cerebellar slices dramatically altered the expression of bivalent genes and induced the downregulation of migration-related genes and upregulation of synaptic genes, inhibited glial-guided migration, and accelerated terminal differentiation. Thus, histone bivalency is required to regulate the timing of the progression from progenitor cells to mature neurons.

Figure 1.

Characterization of gene expression and chromatin landscape in developing mouse cerebellar granule cells. A Schematic of postnatal GC development. During late embryogenesis and the first postnatal week, granule cell progenitors (GCPs) undergo clonal expansion in the external granule cell layer (EGL). Upon exiting the cell cycle, immature GCs extend parallel fibers and a leading process and begin glial-guided migration along Bergmann glia fibers. After passing the Purkinje cell layer (PL), the GCs stop migrating and extend dendrites, forming synapses with mossy fiber afferents to form the cerebellar circuitry. IGL, internal granule layer; ML, molecular layer; P, postnatal day. B Genome browser view of representative genes undergoing gene expression and histone modification changes during GC development. Representative ChIP-seq and TRAP RNA-seq samples are shown. H3K4me3 (n=2–3 samples/group) and H3K27me3 (n=4–5 samples/group) ChIP-seq was performed on chromatin isolated from GCPs (P7) or from GC nuclei sorted using FANS (P12 and P21). RNA-seq was performed on TRAP RNA isolated from P5-P7 Tg(Atoh1-Egfp-L10a) GCPs (P7-GCP, n=4 samples/group), or from the cerebellar lysates of Tg(Neurod1-Egfp-L10a) mice at P7 (P7-GC), P12 and P21 (n=5 mice/group). C Left: Heatmap depicting differentially expressed (DE) genes in developing GCs. DE genes (p-adj < 0.01, log₂FC≥1) were identified by pairwise comparisons between groups using DESeq2 and sorted by the highest expressed genes at each age. Right: Gene Ontology (GO) analysis of the highest expressed genes at each developmental time point, identified using clusterProfiler. The GO biological process categories were sorted by the adjusted P-value and the top 5 enriched non-redundant categories are shown for each age. D Relative signal of the histone modifications H3K4me3 and H3K27me3 around transcription start sites (TSS) at P7, P12 and P21, grouped into quintiles based on gene expression levels.

Figure 2.

H3K4me3/H3K27me3 bivalent promoters are prevalent in proliferating GC progenitors. A Genomic distribution of bivalent peaks. Bivalent peaks were defined as regions where H3K4me3 and H3K27me3 peaks overlapped. The genomic distribution of the identified overlapping regions, defined as bivalent, was determined using the ChIPseeker package. B Enrichment map depicting ChIPseq normalized reads in P7 GCPs, centered at TSS ± 2.5 kb, sorted by H3K4me3 signal, and grouped by TSS status (H3K4me3-only, bivalent, or H3K27me3-only). C Genome browser view or representative bivalent genes. Shaded areas denote the bivalent regions around the TSS. Note that Ccnd1 and Kcna1 are both bivalent at P7 but during migration and maturation Ccnd1 retains H3K27me3 and Kcna1 retains H3K4me3 with a corresponding change in gene expression. Cxcl12 is bivalent at P7 and P21, while H3K27me3 is transiently removed at P12, corresponding to a peak in its expression at P12. D Venn diagrams depicting the number of bivalent, H3K4me3-only, and H3K27me3-only genes across GC development. Examples of bivalent genes are shown. Note that bivalent genes are particularly common in P7 GCPs, while the majority of H3K4me3-only and H3K27me3-only genes are stable between P7 and P21. E Alluvial plot showing the dynamics of histone post-translational modifications between P7 and P21. Bars represent PTM statuses at promoters and lines indicate the changes in PTMs over development. Only genes that are either bivalent or H3K27me3-only at any age are shown. Bivalent genes are highlighted. Groups that contain fewer than 0.5% of included genes are omitted for simplicity. Note that a major fraction of P7 bivalent genes become H3K4me3-only by P12 and remain so through P21, whereas most P7 H3K27me3-only genes remain H3K27me3-only until P21.

Figure 3.

Identification of bivalent domains ex vivo. A Schematic of the cMAP3 bivalency probe used to detect truly bivalent domains ex vivo. The probe consists of a fluorophore (Emerald) fused with H3K27me3 and H3K4me3 reader domains. The plasmid co-expresses tdTomato under IRES for assessing GC morphology. B Representative images of GCs electroporated with the cMAP3 bivalency probe, showing the subnuclear localization of bivalent domains. Proliferating GCPs, imaged 6–12h after electroporation, contain multiple fluorescent foci per nucleus, indicative of bivalent domains. Postmitotic GCs, imaged 48–72h after electroporation, contain no foci. Scale bar, 5 μm.

Figure 4.

H3K4me3/H3K27me3 bivalency predicts gene expression levels in GCs. A Enriched GO (biological process) categories of bivalent genes, identified using the clusterProfiler package. Top 20 identified categories were plotted using the treeplot function to visualize the relationship between the categories. B. At P7, bivalent genes are expressed at lower levels than H3K4me3-only genes but at higher levels than H3K27me3 or no-peak genes. Kruskal-Wallis one-way ANOVA, followed by Dunn’s post hoc test. **** p < 0.0001 between all comparisons. C The expression of bivalent genes correlates with H3K4me3/H3K27me3 ratio at the TSS. One-way ANOVA, followed by Tukey HSD post hoc test. Correction for multiple comparisons was performed considering all comparisons but only significances between adjacent groups are shown. **** p < 0.0001. D Changes in the PTM status of bivalent genes correlate with gene expression across GC development. Pairwise t-test, adjusted for multiple comparisons using the BH method. ** p < 0.01, **** p < 0.0001. E Enriched GO (biological process) categories among genes that change from bivalent to monovalent between P7 and P21, identified with clusterProfile.

Figure 5.

Transcription factors that establish GC identity are bivalent. A Heatmap depicting the developmental gene expression dynamics of selected transcription factors with bivalent promoters. Yellow asterisks denote the time points when the genes are bivalent. B P7 H3K4me3 peaks ranked by width in base pairs. The dashed line denotes the cut-off point between ‘broad’ and ‘typical’ peaks, determined using the elbow method. Peaks annotated to the promoters of bivalent TF genes are shown in blue. C Integrative Genome Browser view of selected transcription factors with bivalent promoters.

Figure 6.

The effect of EZH1/2 inhibition on gene expression in cultured GCs. A Developmental expression of H3K27 methyltransferases Ezh1 and Ezh2, based on TRAP RNAseq (n=4–5/group). B Immunoblot detection of H3K27me3 levels in GCs cultured for 5 DIV in the presence of vehicle control (DMSO) or increasing concentrations of EZH1/2 inhibitor UNC1999. H3 was used as a loading control. C Heatmap depicting differentially expressed genes in response to EZH1/2 inhibition after treatment with 100 nM UNC1999, compared to DMSO. D The percent of genes in each PTM category (based on P7 GCP ChIPseq) that were up- or downregulated in cultured GCs in response to UNC1999 treatment, compared to DMSO. More than 10% of all bivalent genes were differentially expressed and the differentially expressed bivalent genes were significantly more likely to be upregulated than downregulated. Statistical analysis indicates the difference between the distribution of up- and downregulated genes between each PTM group. Pairwise Fisher test was performed using the rstatix package and p-values were corrected for multiple comparisons using the fdr method. ** p < 0.01, *** p < 0.001, **** p < 0.0001. E-G Volcano plots depicting differentially expressed bivalent (E), H3K4me3-only (F), or H3K27me3-only (G) genes in response to UNC1999 treatment, compared to DMSO. The number of up- or downregulated genes is indicated at the top of the chart. H Genome browser representation of RNAseq tracks for example bivalent genes at DIV5. Actb is shown as a control. I Volcano plot depicting differentially expressed genes (p-adjusted < 0.05, indicated with red) in response to UNC1999 treatment, compared to DMSO. Upregulated genes include several well-known marker genes of mature GCs, such as Gabra6, Cbln3, and Grm4. J Enriched GO categories of up- and downregulated genes after UNC1999 treatment compared to DMSO, identified using clusterProfiler package. The GO biological process categories were sorted by the adjusted P-value and the top 5 enriched non-redundant categories are shown for each group.

Figure 7.

H3K4me3/H3K27me3 bivalency regulates the speed of GC neuronal maturation A Schematic of the experimental design. A plasmid encoding the Venus fluorophore was electroporated into the cerebellum and organotypic cerebellar slices were made and cultured in the presence of vehicle control (DMSO) or 200 nM UNC1999 for 60h. The morphology and subcortical localization of GCs was evaluated to identify GCs at different stages of development. B Images of electroporated GCs in ex vivo cerebellar slices, treated with vehicle control (DMSO) or UNC1999 for 60h. Control cells exhibit a bipolar morphology typical of migrating GCs, with a leading process oriented in the direction of migration (white arrows) and few multipolar cells (white asterisks). By contrast, cells treated with UNC1999 displayed inhibited migration and multipolar morphology. Scale bar, 25 μm. C GC migration distance after vehicle control (DMSO) or UNC1999 treatment, measured as the distance of the cell soma to the outer edge of the parallel fiber axons. Each replicate included slices from 2–3 postnatal P8 mouse pups per condition. GC migration distance was quantified from 4 replicates for a total of 868 cells (DMSO) or 2334 cells (UNC1999). D Quantification of the percentage of bipolar and multipolar GCs out of all Venus-expressing cells in the molecular layer. Each replicate included slices from 2–3 postnatal P8 mouse pups per condition. For the vehicle control (DMSO) condition, regions of interest were imaged from 5 replicates for a total of 593 cells. For the UNC1999 condition, regions of interest were imaged from 4 replicates for a total of 880 cells. Student’s t-test. **** p < 0.0001.