Abstract
BackgroundEpigenetic modifications have a central role in transcriptional regulation. While several studies using next-generation sequencing have revealed genome-wide associations between epigenetic modifications and transcriptional states, a direct causal relationship at specific genomic loci has not been fully demonstrated, due to a lack of technology for targeted manipulation of epigenetic modifications. Recently, epigenome editing techniques based on the CRISPR-Cas9 system have been reported to directly manipulate specific modifications at precise genomic regions. However, the number of editable modifications as well as studies applying these techniques in vivo is still limited.ResultsHere, we report direct modification of the epigenome in medaka (Japanese killifish, Oryzias latipes) embryos. Specifically, we developed a method to ectopically induce the repressive histone modification, H3K27me3 in a locus-specific manner, using a fusion construct of Oryzias latipes H3K27 methyltransferase Ezh2 (olEzh2) and dCas9 (dCas9-olEzh2). Co-injection of dCas9-olEzh2 mRNA with single guide RNAs (sgRNAs) into one-cell-stage embryos induced specific H3K27me3 accumulation at the targeted loci and induced downregulation of gene expression.ConclusionIn this study, we established the in vivo epigenome editing of H3K27me3 using medaka embryos. The locus-specific manipulation of the epigenome in living organisms will lead to a previously inaccessible understanding of the role of epigenetic modifications in development and disease.
Highlights
Epigenetic modifications have a central role in transcriptional regulation
We selected promoter regions of 7 genes, Arhgap35, Pfkfb4a, Nanos3, Dcx, Tbx16, Slc41a2a and Kita as targets, because they showed low H3K27me3 enrichment at the blastula stage (Figs. 1c, g, k, n, 2a, d, 3f ). These target promoters do not show any particular (See figure on page.) Fig. 1 H3K27me3 epigenome editing by dCas9-Oryzias latipes H3K27 methyltransferase Ezh2 (olEzh2) targeting hypomethylated promoters. a Schematic of dCas9, dCas9-olEzh2 and dCas9-olEzh2(∆SET) constructs and H3K27me3 induction caused by dCas9-olEzh2. b Schematic view of the dCas9-olEzh2 epigenome editing and injection experiments. single guide RNA (sgRNA) and mRNA were injected at the one-cell stage
RT-qPCR was performed using the pre-early gastrula embryos, because zygotic genome activation (ZGA) occurs at the late blastula in medaka. c, g, k, n The epigenetic modification patterns around Arhgap35, Kita, Nanos3 and Dcx, sgRNAs and ChIP-qPCR product positions
Summary
While several studies using next-generation sequencing have revealed genome-wide associations between epigenetic modifications and transcriptional states, a direct causal relationship at specific genomic loci has not been fully demonstrated, due to a lack of technology for targeted manipulation of epigenetic modifications. The number of editable modifications as well as studies applying these techniques in vivo is still limited Epigenetic modifications, such as histone modifications and DNA methylation, alter gene transcriptional states, thereby regulating various biological processes (e.g., development, cell differentiation and diseases) [1,2,3]. Fukushima et al Epigenetics & Chromatin (2019) 12:17 of transcriptional repression [8, 9]; PRC1 recruitment can subsequently cause PRC2 protein binding in certain genomic regions [10, 11], and at previously active genes, inhibition of transcription results in the recruitment of PRC2 and accumulation of H3K27me3 [12]. Direct manipulation of H3K27me at individual genomic loci is required to fully understand the mechanism of H3K27me3-associated repression
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