dCas9-olEzh2 injection in medaka results in site-specific accumulation of H3K27me3 in vivo
In order to make a new construct for in vivo H3K27me3 manipulation by dCas9 epigenome editing, we first cloned the Oryzias latipes H3K27 methyltransferase Ezh2 (olEzh2) sequence and compared it with human, mouse and zebrafish Ezh2 sequences. The alignment revealed that Ezh2 is highly conserved (98%) among the vertebrate species, especially the CXC domain and the SET domain (100%), which are required for H3K27 methyltransferase activity (Additional file 1: Fig. S1).
To test the ability of olEzh2 to induce H3K27me3 site specifically in vivo, full-length olEzh2 was fused to dCas9 with a FLAG tag at the N-terminus (Fig. 1a). To select target genome regions for H3K27me3 manipulation, we investigated our published ChIP-seq data from medaka blastula embryos [27]. 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 characteristics in terms of CpG contents compared to others. sgRNAs were designed to target DNase I hypersensitive sites using DNase I-seq data from medaka blastula [28], because previous genome-wide Cas9 binding studies showed that chromatin inaccessibility prevents sgRNA/Cas9 complex binding [29, 30]. We used a set of sgRNAs targeting a single promoter region because previous studies showed that multiple sgRNAs at each target promoter increased the efficiency of epigenome editing [17, 31, 32].
We injected dCas9 or dCas9-olEzh2 mRNA along with three or four sgRNAs into medaka, the one-cell-stage (stage 2) embryos, and to examine the recruitment of dCas9 or dCas9-olEzh2 and accumulation of H3K27me3 at the target regions, we performed ChIP-qPCR at the late blastula (stage 11), when histone modifications have already been accumulated after epigenetic reprogramming [27, 33] (Fig. 1b). For each target promoter, several primer pairs that overlap with sgRNAs were designed for ChIP-qPCR. The positive and negative controls for ChIP experiments are described in Additional file 1: Fig. S2. The results of ChIP-qPCR using anti-FLAG antibody confirmed that dCas9-olEzh2 was recruited specifically to the target sites (Figs. 1d, h, l, o, 2b, e, 3g). Importantly, at Arhgap35, Pfkfb4a, Nanos3, Dcx and Kita loci, the level of H3K27me3 increased in dCas9-olEzh2 injected embryos, as compared to non-injected and dCas9 injected ones (Figs. 1e, i, m, p, 3h), demonstrating that dCas9-olEzh2 is capable of inducing site-specific H3K27me3 in vivo. On the other hand, at Tbx16 and Slc41a2a loci, there was no significant induction of H3K27me3 (Fig. 2c, f), even though dCas9-olEzh2 was recruited to the target site (Fig. 2b, e). We hypothesized that some factors were preventing the accumulation of H3K27me3 at these two loci. Analysis of published whole-genome bisulfite sequencing data from medaka blastula embryos [34] revealed that Arhgap35, Pfkfb4a, Nanos3, Dcx and Kita promoters are hypomethylated (Figs. 1c, g, k, n, 3f), whereas Tbx16 and Slc41a2a promoters are highly methylated (Fig. 2a, d). Antagonism between DNA methylation and H3K27 methylation was previously reported in mouse embryonic stem cells [35] and neural stem cells [36] and also in medaka blastula embryos [27], and therefore, preexisting DNA methylation might have inhibited the induction of H3K27me3 by dCas9-olEzh2 at Tbx16 and Slc41a2a promoters.
Since the antagonism between H3K27me3 and H3K27ac has also been reported [37], we further checked whether the level of H3K27ac was affected by the dCas9-olEzh2-induced H3K27me3 accumulation. However, ChIP-qPCR using anti-H3K27ac antibody at the Arhgap35 promoter in the sgArhgap35/dCas9-olEzh2 injected embryos showed no significant differences (Additional file 1: Fig. S3), suggesting that the level of H3K27me3 induced by dCas9-olEzh2 was not sufficient for a detectable level of H3K27ac reduction.
Induced H3K27me3 strengthens site-specific gene repression
Next, we examined whether the induction of H3K27me3 by dCas9-olEzh2 has the function to repress the expression of targeted genes, as H3K27me3 induced by Ezh2 is known as a repressive histone modification [6, 13]. To investigate the repression capacity of dCas9-olEzh2, we chose the zygotically transcribed genes, Arhgap35, Pfkfb4a and Kita, among the five targets that showed H3K27me3 induction. We injected dCas9-olEzh2 mRNA along with sgRNAs targeting the Arhgap35, the Pfkfb4a or the Kita promoter, and performed RT-qPCR at the pre-early gastrula stage (stage 12) (Fig. 1b), which follows the zygotic genome activation (ZGA) at the late blastula stage (stage 11) [38]. As a result, both dCas9- and dCas9-olEzh2-injected embryos showed downregulation of Arhgap35, Pfkfb4a or Kita compared to non-injected ones (Figs. 1f, j, 3i), and this agrees with a previous report indicating that dCas9 itself can interfere with transcriptional elongation, RNA polymerase binding or transcription factor binding [17]. Importantly, the expression of Arhgap35 and Kita in dCas9-olEzh2-injected embryos was significantly lower than that in dCas9-injected ones (Figs. 1f, 3i), suggesting that H3K27me3 have strengthened the repression. On the other hand, the expression level of Pfkfb4a did not show significant difference between dCas9- and dCas9-olEzh2 injected embryos (Fig. 1j). Thus, the effect of H3K27me3 accumulation to gene expression may be different between genes or the levels of H3K27me3 accumulation at Pfkfb4a promoter was too low (Fig. 1i).
To validate that the H3K27me3 deposition is causative of transcriptional repression of target genes, we generated a SET domain-deleted mutant dCas9-olEzh2(∆SET) (Fig. 1a). First, we confirmed that this construct had no ability to induce H3K27me3 at target sites (Fig. 3a, b, g, h). Then, we found that the expressions of the two target genes, Arhgap35 and Kita, were significantly lower in dCas9-olEzh2 injected embryos than in dCas9 or dCas9-olEzh2(∆SET)-injected ones (Fig. 3c, i). To further test the possibility that transcriptional interference by dCas9 complex caused the H3K27me3 deposition [8, 9], we increased the molecular concentration of dCas9-olEzh2(∆SET) up to 550 nM and inhibited the gene expression at the same level as dCas9-olEzh2 injection. (Note that all other experiment in this paper used 350 nM concentration.) Under this condition, dCas9-olEzh2(∆SET) (550 nM)-injected embryos showed strong reduction in transcription of the targeted gene (Fig. 3d). However, neither dCas9-olEzh2(∆SET) (350 nM)-injected embryos nor dCas9-olEzh2(∆SET) (550 nM)-injected embryos showed the accumulation of H3K27me3 at the target region (Fig. 3e). Thus, we concluded that the deposition of H3K27me3 was caused by the enzymatic activity of dCas9-olEzh2, but not by transcriptional interference.
H3K27me3 epigenome editing by dCas9-olEzh2 is highly site-specific
Finally, to globally confirm the specificity of H3K27me3 epigenome editing by dCas9-olEzh2, we performed ChIP-seq of dCas9-olEzh2 mRNA/sgArhgap35 sgRNA-injected or dCas9-olEzh2(∆SET) mRNA/sgArhgap35 sgRNA-injected late blastula (stage 11) embryos using anti-FLAG antibody and anti-H3K27me3 antibody. First, we confirmed that two biological replicates showed consistent distribution of dCas9 binding and H3K27me3 (Additional file 1: Fig. S4a, S4b, S4c, S4d). Thus, in the following analyses, we pooled two replicates. Next, we confirmed the specificity of dCas9-olEzh2(∆SET) and dCas9-olEzh2 recruitment to the target site (Fig. 4a, b, S4a, S4b, S5a, S5b). Finally, we observed that H3K27me3 was only induced at the sgRNA target region in dCas9-olEzh2-injected embryos, while there was no deposition of H3K27me3 in dCas9-olEzh2(∆SET)-injected embryos. Among all H3K27me3 peaks in dCas9-olEzh2(∆SET)- and dCas9-olEzh2-injected embryos, only H3K27me3 enrichment of the sgRNA target region was significantly changed (Fig. 4c, d). These data demonstrate the high specificity of H3K27me3 epigenome editing by dCas9-olEzh2.