DNA methylation-mediated expression of zinc finger protein 615 affects embryonic development in Bombyx mori
摘要: 细胞分裂和分化是胚胎从受精卵发育成多细胞个体的重要步骤，DNA甲基化通过调控基因表达而影响胚胎发育的过程。然而，昆虫DNA甲基化调控胚胎发育的机制仍不清楚。该文研究了DNA甲基化对家蚕早期胚胎的影响。早期胚胎的DNA甲基化组和转录组分析表明，5-甲基胞嘧啶（5mC）主要发生在蛋白质代谢相关基因的基因体5’端区域。DNA甲基转移酶1（Dnmt1）催化转录因子锌指蛋白615（ZnF615）基因的甲基化修饰，从而使ZnF615表达上调，并结合到蛋白质代谢相关基因而调控这些基因的表达。Dnmt1 RNAi显示DNA甲基化介导ZnF615调控了非甲基化修饰的胚胎发育所需的营养代谢相关基因的表达。卵巢和胚胎中的ZnF615基因的5mC位点相同。与Dnmt1敲除类似，敲除ZnF615也可以降低家蚕的胚胎孵化率和卵巢的产卵数。对ZnF615基因甲基化率的分析表明，亲本的卵巢和后代胚胎中的ZnF615基因的5mC位点相同，但5mC比率加倍。因此，该研究结果证明了Dnmt1促进转录因子ZnF615的基因内DNA甲基化，增强了其表达而确保了家蚕卵巢和胚胎发育。Abstract: Cell division and differentiation after egg fertilization are critical steps in the development of embryos from single cells to multicellular individuals and are regulated by DNA methylation via its effects on gene expression. However, the mechanisms by which DNA methylation regulates these processes in insects remain unclear. Here, we studied the impacts of DNA methylation on early embryonic development in Bombyx mori. Genome methylation and transcriptome analysis of early embryos showed that DNA methylation events mainly occurred in the 5' region of protein metabolism-related genes. The transcription factor gene zinc finger protein 615 (ZnF615) was methylated by DNA methyltransferase 1 (Dnmt1) to be up-regulated and bind to protein metabolism-related genes. Dnmt1 RNA interference (RNAi) revealed that DNA methylation mainly regulated the expression of nonmethylated nutrient metabolism-related genes through ZnF615. The same sites in the ZnF615 gene were methylated in ovaries and embryos. Knockout of ZnF615 using CRISPR/Cas9 gene editing decreased the hatching rate and egg number to levels similar to that of Dnmt1 knockout. Analysis of the ZnF615 methylation rate revealed that the DNA methylation pattern in the parent ovary was maintained and doubled in the offspring embryo. Thus, Dnmt1-mediated intragenic DNA methylation of the transcription factor ZnF615 enhances its expression to ensure ovarian and embryonic development.
Figure 1. Effect of Dnmt1 knockout on embryonic development and egg hatching in B. mori
A: Schematic of nucleic acid base deletion site in Dnmt1. B: Functional domain (top) and western blot (bottom) analysis of Dnmt1 protein in wild-type (WT) and Dnmt1-/- mutant. C: Changes in hatching rate of WT and Dnmt1-/- mutant. Each point represents the embryo hatching rate of female WT or Dnmt1-/- silkworms. D: Morphology and structure of WT and Dnmt1-/- embryos. Significant differences were determined by t-test (*: P<0.05; **:P<0.01; ***: P<0.001).
Figure 2. Identification of DNA methylation-modified genes affecting embryonic development in B. mori
A: qRT-PCR (top) and dot blot (bottom) analyses of methyltransferase (Dnmt1) expression levels and 5mC levels at six different embryonic development stages. B: Distribution of mC methylation in CG, CHG, and CHH contexts (H represents A, C, or T) in different gene regions (gene body, exon, intron, and 2 kb upstream and downstream of gene body). C: mCG levels in genes with different expression levels. Genes were classified into four groups based on mean expression levels (High expression: FPKM>100; Middle expression: 10<FPKM≤100; Low expression: FPKM<10; No expression: FPKM<1). D: Volcano plot analysis of differentially expressed genes (DEGs) after Dnmt1 RNAi at blastoderm stage (top). Venn diagram comparing DEGs identified by RNA-seq after Dnmt1 RNAi and methylation genes identified by WGBS in embryos (bottom). E: KEGG analysis of DEGs after Dnmt1 RNAi in B. mori embryos. F: Hierarchical clustering heat map of Dnmt1 and 23 genes both methylated and differentially expressed in (D) during the six different stages of embryo development (fertilized egg, blastoderm, germ-band, organogenesis, reversal period, and head pigmentation).
Figure 3. DNA methylation of ZnF615 by Dnmt1 affects B. mori embryonic development
A: Analysis of functional domain of ZnF615 protein. B: Changes in hatching rate after ZnF615 dsRNA treatment. dsRNA was injected into B. mori embryos within 2 h post-oviposition. Dn: n-Day of embryonic development. C: Change in ZnF615 mRNA levels after Dnmt1 RNAi in B. mori embryos. D: Changes in mCG levels in upstream 2 kb, gene body, and downstream 2 kb regions of ZnF615 gene in B. mori embryos after Dnmt1 RNAi. Significant differences were determined by t-test (*: P<0.05; **: P<0.01).
Figure 4. Effect of DNA methylation in promoter and gene body on transcription of ZnF615 gene
A: Schematic of eight 100 bp fragments with the largest differences in mCG levels in promoter and gene body of ZnF615 gene after Dnmt1 RNAi. B: Effects of different promoter (top) and gene body (bottom) regions on luciferase activity. Different regions (regions 1–4) of promoter fragments were inserted upstream of the actin promoter and luciferase ORF. Different regions (regions 5–8) of the gene body were constructed into the luciferase vector between the actin promoter and luciferase ORF. C: Changes in activity of luciferase reporters containing WT or cytosine-mutated region 5 (top) or 8 (bottom) of the gene body of ZnF615 in Bm12 cells co-transfected with dsDnmt1 or dsGFP. Significant differences were determined by t-test (*: P<0.05; **: P<0.01).
Figure 5. EMSA analysis of Dnmt1 binding with regions 5 and 7 in ZnF615 gene body
A: Binding of nuclear proteins isolated from B. mori embryos with region 5 probe. B: Binding of nuclear proteins isolated from B. mori embryos with region 7 probe. Cold probe is unlabeled region 5 or 7 oligos. C: Supershifted band was detected using anti-Dnmt1 antibody. IgG was used as a negative control. Sequences of WT and mutant region 5 and 7 probes are shown in Supplementary Table S9.
Figure 6. Loss-of-function analysis of ZnF615 in B. mori embryos
A: Expression patterns of Dnmt1 and ZnF615 in different tissues, including epidermis, silk gland, testis, ovary, gut, wing disc, and fat body (top), and at different developmental stages, including embryo, 1st–5th instar larval, wandering larval, pupal, and adult stages (bottom). B: Schematic of nucleic acid base deletion site in ZnF615 (top). C: Functional domain analysis of ZnF615 protein in WT and ZnF615-/- mutant. D: Western blot analysis of ZnF615 protein in WT and ZnF615-/- mutant embryos (top). Hatching rates in WT and ZnF615-/- mutant (bottom). Each point represents an embryo hatching rate of female WT or ZnF615-/- silkworms. E: Morphology and structure of WT and ZnF615-/- mutant embryos. Significant differences were determined by t-test (*: P<0.05; **: P<0.01; ***: P<0.001).
Figure 7. DAP-seq analysis of ZnF615 in B. mori embryos
A: Overview of DAP-seq experimental process. cDNA of ZnF615 ORF fused to Halo affinity tag was expressed in vitro and recombinant protein was bound to ligand-coupled beads. Genomic DNA at blastoderm stage of B. mori embryo was ultra-sonicated to 200 bp fragments, which were ligated with Illumina-based sequencing adaptors. HaloTag-ZnF615 protein was then incubated with adapter-ligated genomic DNA library. After unbound DNA fragments were washed away, the ZnF615-bound fragments were released. Released DNA fragments were then purified and sequenced. B: Peak frequency in different gene body regions between 2 kb upstream and downstream. C: Top-ranked motif in ZnF615 DAP-seq data was TTTTTATTGTTTTT. TSS: transcription start site; TTS: transcription termination site. Motifs were determined by MEME analysis using top-ranked peaks. D: Venn diagram comparing DEGs identified by RNA-seq after Dnmt1 RNAi and ZnF615 binding genes by DAP-Seq at blastoderm stage of embryo (top). Change in mRNA levels of genes both peak related and differentially expressed after Dnmt1 RNAi in ZnF615 RNAi B. mori embryos (bottom). E: Changes in mRNA levels of DEGs related to nutrient metabolism pathways enriched by Dnmt1 RNAi in ZnF615 RNAi B. mori embryos. F: Analysis of mCG levels and regions of ZnF615 in B. mori ovary and blastoderm stage of embryo. Significance of results was determined by t-test (*: P<0.05; **: P<0.01).
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