Identification of chromatin states during zebrafish gastrulation using CUT&RUN and CUT&Tag

Abstract

Background: Cell fate decisions are governed by interactions between sequence-specific transcription factors and a dynamic chromatin landscape. Zebrafish offer a powerful system for probing the mechanisms that drive these cell fate choices, especially in the context of early embryogenesis. However, technical challenges associated with conventional methods for chromatin profiling have slowed progress toward understanding the exact relationships between chromatin changes, transcription factor binding, and cellular differentiation during zebrafish embryogenesis.

Results: To overcome these challenges, we adapted the chromatin profiling methods Cleavage Under Targets and Release Using Nuclease (CUT&RUN) and CUT&Tag for use in zebrafish and applied these methods to generate high-resolution enrichment maps for H3K4me3, H3K27me3, H3K9me3, RNA polymerase II, and the histone variant H2A.Z using tissue isolated from whole, mid-gastrula stage embryos. Using this data, we identify a subset of genes that may be bivalently regulated during both zebrafish and mouse gastrulation, provide evidence for an evolving H2A.Z landscape during embryo development, and demonstrate the effectiveness of CUT&RUN for detecting H3K9me3 enrichment at repetitive sequences.

Conclusions: Our results demonstrate the power of combining CUT&RUN and CUT&Tag methods with the strengths of the zebrafish system to define emerging chromatin landscapes in the context of vertebrate embryogenesis.

Keywords: CUT&RUN; CUT&Tag; chromatin; gastrulation; zebrafish.

Figure 1:. CUT&RUN detects H3K4me3 and H3K27me3 at the transcriptional start sites of annotated genes in shield stage embryos.

(A) Heat maps of replicate data for H3K4me3 (green) and H3K27me3 (purple) enrichment as detected by CUT&RUN. (B) Profile plot of mean enrichment of H3K4me3 and H3K27me3 at the TSS of annotated genes. (C) Genome browser view of H3K4me3 and H3K27me3 enrichment at select loci. H3K4me3 is detected at the TSS of three genes with associated RNA transcripts (alkbh6, capns1a, and zgc153990) and H3K27me3 is detected at the TSS of the inactive dlb gene. (D) Genome browser view of broad H3K27me3 enrichment over the hoxA gene cluster on chromosome 3. In C and D replicate data are shown as individual tracks. Rep= Replicate.

Figure 2.. Regions marked with both H3K4me3 and H3K27me3 are detected in chromatin isolated from whole shield stage zebrafish embryos.

(A) Venn diagram indicating the number of regions enriched in H3K4me3, H3K27me3 or both modified histones in CUT&RUN data from whole shield stage embryos. Regions where one peak of H3K27me3 overlapped more than one peak of H3K4me3, or vice versa, were counted as one overlapping peak. (B) Genome browser view illustrating the co-occurrence of H3K4me3 and H3K27me3 enrichment peaks at the TSS of the igfbp2a gene, which lacks detectable transcripts at shield stage. At the same stage, the TSS of the nearby tra2b gene is exclusively marked by H3K4me3 and is associated with RNA transcripts. Replicate data are shown as individual tracks. (C) GO analysis reveals genes marked by both H3K4me3 and H3K27me3 at shield stage are associated with developmental processes including nervous system development (3831 query terms). Rep= Replicate.

Figure 3.. CUT&RUN detects Ser-5 CTD phosphorylated RNA polymerase 2 (Ser5P-pol II) at the TSS of transcribed genes in shield stage embryos.

(A) Heat maps of replicate data for Ser5P-pol II enrichment at the TSS of annotated genes (B) Profile plot showing mean enrichment of Ser5-pol II at the TSS of annotated genes. (C) Genome browser view showing enrichment of Ser5-pol II at the start of setdb1a, which produces RNA transcripts at shield stage. Similar S5-pol II enrichment is not observed at the silent si:ch211-81a5.5 gene. Replicate data are shown as individual tracks. Rep= Replicate.

Figure 4.. CUT&RUN detects H3K9me3 enrichment at repeated sequences in shield stage embryos.

(A) Heat map of H3K9me3 enrichment around peak centers in CUT&RUN replicates (B) Pie chart depicting H3K9me3 enrichment at different repeat sequence classes as detected by CUT&RUN. (C) Venn diagram showing shared and unique peaks between shield stage CUT&RUN and ChIP data. (D) Genome browser view comparing enrichment and called peaks in CUT&RUN (dark blue) and ChIP (light blue) data from shield stage embryos. (E) Genome browser view of a select genomic loci containing a ChIP-only peak. (F) Genome browser view of a select genomic loci containing multiple CUT&RUN-only peaks on the long arm of chromosome 4. (G) Bar graph of CUT&RUN-only, shared and ChIP-only peaks detected in regions with mappability scores below 0.5 (H) Bar graph comparing CUT&RUN-only and ChIP-only peaks on the long vs short arms of chromosome 4. In D-F replicate data are shown as individual tracks. Rep= Replicate.

Figure 5.. CUT&Tag detects H2A.Z in shield stage zebrafish embryos.

(A) Heatmaps of replicate data for H2A.Z enrichment at annotated genes as detected by CUT&Tag in shield stage embryos. (B) Profile plot of H2A.Z mean enrichment at annotated genes. (C) Genome browser views of H2A.Z and RNA transcripts at selected loci. Replicate data for H2A.Z CUT&Tag are shown as individual track. Rep= Replicate.

Figure 6.. CUT&Tag identifies changes of H2A.Z patterning from shield stage to 24 hpf zebrafish embryos.

(A) Correlation heatmap demonstrating high correlation among CUT&Tag replicates and separation based on developmental stages. (B) Heatmaps of H2A.Z enrichment at H2A.Z-marked regions as detected by CUT&Tag in shield stage and 24 hpf embryos. Normalized H2A.Z signals were generated using merged replicate data. (C) Profile plot of H2A.Z enrichment at H2A.Z-marked regions in shield stage and 24 hpf embryos. (D) Venn diagram showing shared and unique H2A.Z peaks between H2A.Z CUT&Tag data in shield stage and 24 hpf embryos. (E) Gene ontology analysis of shield stage and 24 hpf unique H2A.Z peaks. Only top 6 non-redundant GO terms were shown. (F) Genome browser views of H2A.Z enrichment in shield stage and 24 hpf embryos at selected loci.

Figure 7.. Similar enrichment profiles between CUT&RUN replicates.

(A) Principal component analysis (PCA) plot of CUT&RUN sample. (B) Heatmap depicting Spearman correlation of enrichment between CUT&RUN samples, correlation coefficients shown for each pairwise comparison. Rep= Replicate.