Methylation profiling by high throughput sequencing Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing
Summary
Unlike that of mammals, the total DNA methylome of many cold-blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish, this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just after fertilization for oocyte, as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles, the zygotic mutant zebrafish of dnmt1, the major DNA methylation maintenance methyltransferase, surprisingly can develop to term. To solve the role of DNA methylation in early development, we generated zebrafish embryos derived from dnmt1-knocking down oocytes using a recently developed method OMIS (Oocyte Microinjection in situ), which successfully eliminated DNA methylation before zygotic genome activation. dnmt1-deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53-regulated apoptosis, likely triggered by the derepression of transposable elements. Single cell RNA-seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly, the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers, which acquire ectopic H3K27ac, accessible chromatin, and H3K4me3. These somatic enhancers are preferentially CG-rich and are bound by CG-containing TFs. By contrast, embryonic enhancers are generally CG-poor, methylation-insensitive, and are bound by CG-less TFs. Hence, the global DNA methylome inheritance is essential for vertebrate early development, and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs.
Overall design
By employing MethylC-seq, total RNA-seq, scRNA-seq, CUT&RUN, ChIP-seq, and ATAC-seq in control and dnmt1 mKD embryos at oocyte, 256-cell, dome and shield stages in zebrafish, we interrogated the function of DNA methylome and its inheritance in early zebrafish development.
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