Loss of the Polycomb group protein Rnf2 results in derepression of tbx-transcription factors and defects in embryonic and cardiac development

Abstract

The Polycomb group (PcG) protein family is a well-known group of epigenetic modifiers. We used zebrafish to investigate the role of Rnf2, the enzymatic subunit of PRC1. We found a positive correlation between loss of Rnf2 and upregulation of genes, especially of those whose promoter is normally bound by Rnf2. The heart of rnf2 mutants shows a tubular shaped morphology and to further understand the underlying mechanism, we studied gene expression of single wildtype and rnf2 mutant hearts. We detected the most pronounced differences at 3 dpf, including upregulation of heart transcription factors, such as tbx2a, tbx2b, and tbx3a. These tbx genes were decorated by broad PcG domains in wildtype whole embryo lysates. Chamber specific genes such as vmhc, myh6, and nppa showed downregulation in rnf2 mutant hearts. The marker of the working myocard, nppa, is negatively regulated by Tbx2 and Tbx3. Based on our findings and literature we postulate that loss of Rnf2-mediated repression results in upregulation and ectopic expression of tbx2/3, whose expression is normally restricted to the cardiac conductive system. This could lead to repression of chamber specific gene expression, a misbalance in cardiac cell types, and thereby to cardiac defects observed in rnf2 mutants.

Figure 1

Zygotic rnf2 mutant zebrafish embryos show a pleiotropic phenotype. (a) Lateral view of wildtype embryos (left panel) and rnf2 mutant embryos (right panel) at 3 dpf. The rnf2 mutants show a pleiotropic phenotype, including motility problems, craniofacial defects (arrowheads), lack of pectoral fins, and a pronounced heart edema (arrowheads). Not all phenotypes are visible in the pictures. Scale bar = 1 mm. (b) Expression of tissue-specific markers was assessed by WISH at 3 dpf in wildtype and rnf2 mutant embryos. fabp2: intestinal marker, fabp10: liver marker, try: exocrine pancreas marker (arrowhead indicates pancreatic lobe), and myl7: cardiomyocyte marker. Scale bar is 200 µm.

Figure 2

Rnf2 has a similar DNA-binding pattern as H3K27me3 and the presence of H3K27me3 mark is retained upon loss of Rnf2. (a) Heatmaps showing k-means clustering of Rnf2 and H3K27me3 ChIP-seq peaks in promoter regions in wildtype embryos and rnf2 mutant embryos at 3 dpf. In the figure 20 kb regions, with a viewpoint around the center of all peaks, are shown. (b) Bandplot showing the intensities of the ChIP-seq peaks for Rnf2 and H3K27me3 in wildtypes and rnf2 mutants at 3 dpf, and the input of the five clusters defined by k-means clustering. The number of peak regions included in each cluster is depicted above the plot. The black line indicates the median, the intense color 50% of the peaks, and the light color 90% of the peaks.

Figure 3

Loss of Rnf2 is directly associated with upregulation of gene expression. (a) MA-plot of differentially expressed genes between wildtypes and rnf2 mutants at 3 dpf. Significantly differentially expressed genes |Log2FC ≥ 1|; padj < 0.1 are highlighted in red. In total 292 genes are upregulated and 200 genes are downregulated. (b) Gene Set Enrichment Analysis (GSEA) for the genes whose promoters belong to the five clusters defined in Fig. 2a. Cluster 1, 2, 4, and 5 show a non-significant distribution of the genes based on their expression changes upon the rnf2 mutation. Genes belonging to the promoters from cluster 3 are enriched for upregulation upon mutation of rnf2. p-value = < 0.001; NES = 2.04. (c) Gene ontology of biological processes analysis of the 112 genes belonging to cluster 3. (d) ChIP-seq and RNA-seq coverage at the lbx1a gene and the tal1 gene in 3 dpf zebrafish embryos. Light blue: Rnf2 ChIP-seq tracks in wildtypes. Teal: Rnf2 ChIP-seq tracks in rnf2 mutants. Orange: H3K27me3 ChIP-seq track in wildtypes. Brown: H3K27me3 ChIP-seq track in rnf2 mutants. Lilac: wildtype input. Green: RNA-seq track in wildtypes. Red: RNA-seq track in rnf2 mutants.

Figure 4

Zebrafish rnf2 mutant embryos show cardiac looping defects. (a) Fluorescent images of wildtype and rnf2 mutant sibling embryos in a Tg(myl7::GFP) background at 3 dpf. Scale bar is 500 µm. (b) Stills of live-imaging by light sheet microscopy of wildtype and rnf2 mutant hearts in a Tg(myl7::GFP) background starting at 1 dpf. Scale bar is 100 µm.

Figure 5

Single HeartsRNA-seq is used to assess transcriptional differences between wildtype and rnf2 mutant hearts over time. (a) Workflow of Single HeartsRNA-seq. Zebrafish hearts were manually dissected at 1, 2, and 3 dpf as described previously. The remaining tissue was used for genotyping and the rnf2 mutant and wildtype hearts were sequenced. (b) Differentially expressed genes |Log2FC > 0|; padj < 0.01 between rnf2 mutant and wildtype hearts are visualized in the bar graphs. Yellow: upregulated (up) in rnf2 mutants compared to wildtypes. Blue: downregulated (dn) in rnf2 mutants compared to wildtypes.

Figure 6

Cardiac chamber identity is disrupted in rnf2 mutants at 3 dpf. (a) GSEA using the list of cardiac genes reported by Hill et al. indicates that the group of transcription factors is significantly enriched in being upregulated in rnf2 mutant hearts at 3 dpf as detected by Single HeartsRNA-seq (FDR q-value = 0.001; NES = 1.96). The leading transcription factors (n = 9) are listed in the zoom of the GSEA plot. (b) GSEA indicates the annotation ‘Structural Group’ genes (n = 23) to be enriched for downregulation upon the rnf2 mutation in hearts at 3 dpf. The leading genes (n = 8) are listed in the zoom of the GSEA plot. FDR q-value = 0.054; NES = −1.67. (c) Normalized counts as found by Single HeartsRNA-seq for nppa, myl7, myh6, and vmhc at 3 dpf in wildtype and rnf2 mutant hearts with their accompanying whole mount in situ hybridizations. Scale bar is 200 µm.