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Abstract
Accurate fractionation of genomes into euchromatin and heterochromatin is essential for successful transcriptional regulation and development. Trimethylation of histone 3 lysine 9 (H3K9me3) is an essential component of heterochromatin, and bulk establishment of H3K9me3 has been widely observed during animal embryogenesis. However, the mechanisms driving this process are not fully understood, especially in early vertebrate development. Zebrafish offer a powerful system to investigate heterochromatin establishment and regulation in embryogenesis. Here, we adapted the chromatin profiling method Cleavage Under Targets and Release Using Nuclease (CUT&RUN) for zebrafish embryos to overcome challenges faced by conventional chromatin profiling methods. Using CUT&RUN, we generate enrichment maps of H3K4me3, H3K27me3, H3K9me3, and RNA polymerase II, identify a subset of bivalently regulated developmental genes, and demonstrate the effectiveness of CUT&RUN for assaying H3K9me3 enrichment at repetitive sequences. We go on to further investigate the genome-wide distribution of H3K9me3 in early development, finding that LTR transposable elements are disproportionately enriched given their low prevalence in the genome. We then generate a high-resolution time course of H3K9me3 CUT&RUN across embryogenesis and discover low amplitude, pericentromeric, H3K9me3 that is detectable before zygotic genome activation and bulk H3K9me3 establishment. To interrogate H3K9me3 targeting mechanisms we knock down the two pairs of H3K9me3 histone methyltransferases in zebrafish, setdb1a/setdb1b and suv39h1a/suv39h1b, and find they differentially target a subset of transposable elements.
We then investigate the divergent functions of Suv39h1 methyltransferases in the early embryo. We show that Suv39h1a, like murine Suv39h2 and human SUV39H1, is highly enriched for basic amino acids in its N-terminus tail – a feature that in mammalian systems has been shown to facilitate localization to the mitotic pericentromeres. We generate suv39h1a and suv39h1b maternal-zygotic zebrafish mutants and find that Suv39h1a, but not Suv39h1b, is responsible for early embryo pericentromeric H3K9me3. suv39h1a maternal zygotic mutants have abnormal nuclei and severe, stochastic developmental defects, likely due to high levels of genomic instability. Taken together, this work improves the detection of H3K9me3 enrichment, establishes a baseline of how H3K9me3 dynamics change over early development, and investigates whether H3K9me3 methyltransferases Suv39h1a and Suv39h1b have divergent functions in the early embryo.