Temporal analysis of enhancers during mouse cerebellar development reveals dynamic and novel regulatory functions
- Centre for Molecular Medicine and Therapeutics, BC Children’s Hospital Research Institute, Canada
- University of British Columbia, Canada
- RIKEN, Japan
- The Centre for Applied Genomics, The Hospital for Sick Children, Canada
Figures
Figure 1 with 1 supplement
Enhancer identification during cerebellar development.
(A) An overview of the stages of cerebellar development profiled in this study. The datasets collected at these ages and the downstream analyses are shown in the flow chart. Labels: NE: …
Figure 1—figure supplement 1
Validation of identified enhancer sequences.
(A) Sample-sample correlation heatmap and dendrogram comparing H3K4me1 and H3K27ac profiles of all samples collected in this study. (B) Bar plot displaying the percentage of H3K27ac peaks collected …
Figure 2 with 4 supplements
Enhancer activity is dynamic throughout cerebellar development.
(A) Volcano plots showing robust cerebellar enhancers with differential H3K27ac peak signal for three comparisons: E12 vs P9, E12 vs P0, and P0 vs P9. Differential signal strength was identified for …
Figure 2—figure supplement 1
Overlap with histone profiles from the developing hindbrain, midbrain and forebrain.
(A–B) Venn diagrams showing overlap of H3K27ac and H3K4me1 peaks called at E12 (A) and P0 (B) in the developing cerebellum with hindbrain, midbrain and forebrain samples at their respective ages. (C)…
Figure 2—figure supplement 2
Donut chart displaying the proportion of robust cerebellar enhancers that were found to overlap with one, two or three brain regions examined (hindbrain, midbrain and forebrain) or found to be active only in cerebellar samples (CB only).
The outer donut chart shows the percentage of Early and Late active enhancers for each category (tissue).
Figure 2—figure supplement 3
Box plots of Early and Late active enhancer activity for robust cerebellar enhancers with overlapping activity in the developing hindbrain, midbrain and forebrain.
Y-axis indicates the H3K27ac ChIP-seq signal in each brain region for Early or Late active robust cerebellar enhancers.
Figure 2—figure supplement 4
Cell type assignment of robust cerebellar enhancers using snATAC-seq as generated by Sarropoulos et al., 2021 in the developing cerebellum.
(A) Donut plot displaying the proportion robust cerebellar enhancers assigned to the cell-types of the developing cerebellum. (B) Bar plot indicating the percentage of Early and Late robust …
Figure 3 with 2 supplements
Neural transcription factors with known and novel function in the developing cerebellum are enriched in dynamic cerebellar enhancers.
(A) Dot plot displaying significantly enriched (adjusted p-value <1E-11) motifs and the predicted matching transcription factor (TF). Displayed are the results for Early (top) and Late (bottom) …
Figure 3—figure supplement 1
Immunofluorescent analysis of Pax3 expression in the developing mouse cerebellum.
(A) Bar plot showing the percentage of Pax3 + cells co-stained (y-axis) with cerebellar cell markers Pax2, Foxp2 and Calbindin at E12, E15, and P3 (x-axis). (B) Top: Immunofluorescent co-staining of …
Figure 3—figure supplement 2
Pax3 expression in the developing mouse and human cerebellum quantified by CAGE-seq and scRNA-seq.
(A) Line plot displaying Pax3 CAGE-seq expression in the developing cerebellum. Y-axis represents normalized expression in transcripts per million (TPM). (B) Bar plots and (C) radar plots displaying …
Figure 4 with 2 supplements
Correlated Early target genes are expressed in spatially distinct areas and have diverse roles in cerebellar development.
(A) Line plot and heatmap showing mean z-score expression for Early target genes throughout the cerebellar time course. (B) Line graph representation of expression pattern throughout time for each …
Figure 4—figure supplement 1
Enrichment analysis of cerebellar genes in robust enhancer target gene list and Elbow anlaysis to determine the optimal k value for k-means clustering.
(A) Histogram plotting the number of permutations (y-axis) of 2064 genes expressed in the cerebellum against the number of cerebellar genes found within a randomly generated list of genes. The red …
Figure 4—figure supplement 2
Putative target genes of robust cerebellar enhancers containing Pax3 DNA binding motifs regulate neural progenitor function.
Left: Gene Ontology (GO) enrichment analysis of all target genes of Early active robust cerebellar enhancers containing Pax3 DNA-binding motifs, displaying the top enriched GO terms. Size of the …
Figure 5
Correlated Late target genes are expressed in developing granule cells or Purkinje cells with common roles in cerebellar development.
(A) Line plot and heatmap showing mean z-score expression throughout the cerebellar time course. (B) Line plot representation of expression pattern throughout time for each cluster. (C) Known …
Figure 6 with 2 supplements
Bhlhe22 is expressed in differentiating granule cells in postnatal cerebellar development.
(A) Bhlhe22 (green) and Neurod1 (red) immunofluorescent co-staining at P9.5 of taken from a posterior lobe IX. (B) Bhlhe22 (green) and NeuN (red) immunofluorescence co-staining at P6 taken from …
Figure 6—figure supplement 1
Transcription factor Bhlhe22 is expressed in the granule cell layer during cerebellar development.
(A) The Bhlhe22 locus (chr3:17,941,033–17,960,000) in IGV showing H3K27ac and H3K4me1 profiles across biological replicates of E12, P0, P9 cerebella. The predicted Bhlhe22 enhancer is highlighted …
Figure 6—figure supplement 2
Bhhe22 expression in the developing mouse and human cerebellum quantified by scRNA-seq.
(A) Bar plots and (B) radar plots displaying mouse (top; Carter et al., 2018) and human (bottom; Aldinger et al., 2021) scRNA-seq average expression of Bhlhe22/BHLHE22 for cell type clusters defined …
Figure 7
Knockdown of Bhlhe22 reduces migration of cultured cerebellar granule cells.
(A) Workflow for dissociated and reaggregate postnatal granule cell cultures. (B) RT-qPCR analysis of Bhlhe22 gene expression in dissociated postnatal granule cell cultures after treatment with …
Figure 8 with 3 supplements
Cerebellar enhancers are enriched for GWAS SNPs and DNMs associated with ASD.
(A) Enrichment analysis of ASD-associated and chronic kidney disease associated (negative control) GWAS variants in cerebellar enhancers and H3K27ac peaks called from E12, P0, and P9 samples. (B) …
Figure 8—figure supplement 1
Integrative Genomics Viewer visualization of a de novo 11 kb deletion overlapping an enhancer predicted to target the CDC42BPB gene.
Evidence supporting the correctness of the deletion in the proband includes the 50% drop in read depth, along with read pairs mapping further apart than expected (red lines). No such evidence was …
Figure 8—figure supplement 2
Sequence alignment between mouse and humans for CDC42BPB and an enhancer element predicted to regulated CDC42BPB expression.
(A) Ensembl browser screenshot depicting the sequence alignment of CDC42BPB between humans and mouse showing a conservation of coding sequence. (B) Ensembl browser screenshot showing sequence …
Figure 8—figure supplement 3
Cdc42bpb expression in the developing mouse and human cerebellum quantified by scRNA-seq.
(A) Bar plots and (B) radar plots displaying mouse (top; Carter et al., 2018) and human (bottom; Aldinger et al., 2021) scRNA-seq average expression of Cdc42bpb/CDC42BPB for cell type clusters …
Tables
Table 1
A list of enhancer-regulated target genes from Late Cluster 1 found to be significantly differentially expressed in the conditional Atoh1 knockout mouse.
The second and third column contain the observed P-value and fold change from the differential expression analysis, respectively. The fourth and fifth columns indicate whether the gene has …
| Gene | p-value (Atoh1-null) | Fold Change (Atoh1-null) | Cerebellar Development | Reference (PMID) |
|---|---|---|---|---|
| Neurod1 | 9.196E-229 | 0.2 | X | 19609565 |
| Nfix | 1.1991E-43 | 0.53 | X | 21800304 |
| Zic1 | 1.3082E-37 | 0.35 | X | 21307096 |
| Barhl1 | 2.2505E-35 | 0.22 | X | 9412507 |
| Zic2 | 2.0968E-20 | 0.34 | X | 11756505 |
| Insm1 | 5.4814E-20 | 0.25 | X | 18231642 |
| Tcf4 | 9.9139E-20 | 0.69 | X | 30830316 |
| Nfia | 1.8675E-16 | 0.6 | X | 17553984 |
| Bhlhe22 | 4.7662E-10 | 0.53 | ||
| Purb | 2.5878E-09 | 0.53 | ||
| Neurod2 | 4.424E-09 | 0.37 | X | 11356028 |
| Klf13 | 1.7938E-06 | 0.72 | ||
| Zfp521 | 3.5899E-06 | 0.8 | X | 24676388 |
| Sox18 | 3.7168E-05 | 0.71 | ||
| Nfib | 0.00021009 | 0.63 | X | 17553984 |
Additional files
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Supplementary file 1
Coordinates in BED file format of all 7024 robust cerebellar enhancers.
- https://cdn.elifesciences.org/articles/74207/elife-74207-supp1-v2.zip
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Supplementary file 2
Coordinates of Early and Late active robust cerebellar enhancers.
- https://cdn.elifesciences.org/articles/74207/elife-74207-supp2-v2.xlsx
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Supplementary file 3
Quality control metrics evaluating H3K27ac and H3K4me1 ChIP-seq sensitivity.
Map%: Percentage reads mapped, Filt%: percentage of reads filtered, Dup%: duplication rate, ReadL: read length, FragL:, FRiP%: fraction of reads in peaks, RSC: relative strand correlation.
- https://cdn.elifesciences.org/articles/74207/elife-74207-supp3-v2.xlsx
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Supplementary file 4
TF motifs enriched in robust cerebellar enhancers.
This table contains the motif symbol, the TF in the JASPAR database that best matches the enriched motif, protein family of the best match TF, percentage of robust cerebellar enhancers and background sequences containing each motif.
- https://cdn.elifesciences.org/articles/74207/elife-74207-supp4-v2.xlsx
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Supplementary file 5
List of the most highly correlated putative gene targets of robust cerebellar enhancers.
This file contains coordinates of the enhancer, gene target symbol, and Pearson correlation coefficient.
- https://cdn.elifesciences.org/articles/74207/elife-74207-supp5-v2.xlsx
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Supplementary file 6
Robust cerebellar enhancers containing a Pax3 motif and their putative target genes.
The rightmost column indicates whether the putative target gene has been previously implicated in cerebellar development.
- https://cdn.elifesciences.org/articles/74207/elife-74207-supp6-v2.xlsx
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Supplementary file 7
Coordinates of mouse robust cerebellar enhancers and putative target genes converted to human genome build hg38.
The rightmost column (‘Orthologous’) indicates whether an enhancer and it’s putative target genes are found on the same chromosome after conversion.
- https://cdn.elifesciences.org/articles/74207/elife-74207-supp7-v2.xlsx
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Supplementary file 8
Gene targets for enhancers enriched with ASD variants from the GREGOR analysis.
The second column indicates the group to which the gene belongs to in the SFARI gene database of ASD candidate genes. S: Syndromic gene category; 1: Category 1 (High Confidence); 2: Category 2 (Strong Candidate).
- https://cdn.elifesciences.org/articles/74207/elife-74207-supp8-v2.xlsx
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Supplementary file 9
Primers used for RT-qPCR analysis.
- https://cdn.elifesciences.org/articles/74207/elife-74207-supp9-v2.xlsx
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Supplementary file 10
GWAS Catalog traits used to identify variants associated with ASD.
All variants associated with these traits were used as input for the ASD variant enrichment analysis conducted using GREGOR.
- https://cdn.elifesciences.org/articles/74207/elife-74207-supp10-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/74207/elife-74207-transrepform1-v2.pdf
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Source code 1
Generate psuedobulk expression values (CPM) by developmental stage.
- https://cdn.elifesciences.org/articles/74207/elife-74207-code1-v2.zip
