Mbd5 interacts with PR-DUB and is necessary and sufficient to promote deubiquitylation. (A) Schematic of the experimental flow of in vivo co-IP and mass spectrometry. (B) A table listing all proteins belonging to the PR-DUB complex identified in LC–MS/MS. (C) Schematic diagram of the PR-DUB complex. (D–G) Representative images of western-blot and quantification of the expression level of H2AK119ub1, H3K27ac and H3K27me3 in mbd5Δ29 mutants (D, E), and in Tg[zhsp70l:FLAGmbd5iso2-E2AGFP] (F-G). H3 was used as the internal control. All data in E and G represent mean ± SEM, ns, no significance, *P< 0.05, **P< 0.01. (H) Enrichment of the Mbd5 CLIP signal (top left), RNA m5C signal (top right), and histone H2AK119ub1 (bottom left) on Mbd5 targets. The Venn diagram depicts the overlap between m5C-marked RNAs and Mbd5-bound RNAs. The density plot (top right) was generated by plotHeatmap function provided by deeptools package, using the Mbd5 CLIP-seq bigWig files and bed files from the published RNA m5C bisulfite sequencing (44). The density plot (bottom left) was generated by plotHeatmap function provided by deeptools package, using the Mbd5 CLIP-seq bigWig files and bed files from H2AK119ub1 ChIP-seq datasets (52). (I) IGV tracks showing DNA 5mC, RNA m5C, and H2AK119ub1 occupancy on an example Mbd5 target (hbae3) and an example non-Mbd5 target (kank2). (J) A model: the zebrafish Mbd5 protein is a mRNA m5C reader. By binding to nascent m5C-modified mRNA, it recruits or stabilizes the PR-DUB complex to promote histone deubiquitylation at H2AK119 sites. Mbd5 thus regulates the transcription of genes required for normal development, metabolism, and behavior.
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