Dnmt1a is essential for gene body methylation and the regulation of the zygotic genome in a wasp

Abstract

Gene body methylation (GBM) is an ancestral mode of DNA methylation whose role in development has been obscured by the more prominent roles of promoter and CpG island methylation. The wasp Nasonia vitripennis has little promoter and CpG island methylation, yet retains strong GBM, making it an excellent model for elucidating the roles of GBM. Here we show that N. vitripennis DNA methyltransferase 1a (Nv-Dnmt1a) knockdown leads to failures in cellularization and gastrulation of the embryo. Both of these disrupted events are hallmarks of the maternal-zygotic transition (MZT) in insects. Analysis of the embryonic transcriptome and methylome revealed strong reduction of GBM and widespread disruption of gene expression during embryogenesis after Nv-Dnmt1a knockdown. Strikingly, there was a strong correlation between loss of GBM and reduced gene expression in thousands of methylated loci, consistent with the hypothesis that GBM directly facilitates high levels of transcription. We propose that lower expression levels of methylated genes due to reduced GBM is the crucial direct effect of Nv-Dnmt1 knockdown. Subsequently, the disruption of methylated genes leads to downstream dysregulation of the MZT, culminating in developmental failure at gastrulation.

Fig 1. Live imaging of control and Nv-dnmt1a RNAi embryos.

A) Box and whisker plot displaying the length of blastoderm division times that were measured during live imaging experiments. Nc = nuclear cycle #. P values were determined by performing a 2-tailed T-test assuming unequal variance between RNAi and control. N = 5 control, N = 5 Nv-dnmt1a RNAi. B-C) Live-imaging of a control embryo at the onset of gastrulation and at post-gastrulation. Peach arrows in (B) point to the initial mesodermal folds which are eventually internalized C. The blue arrowhead in C points to the serosa. Scale bars (black lines in B and C) are 50μm. (D-E) Live-imaging of a Nv-dnmt1a RNAi embryo at the onset of gastrulation and at what is equivalent to post-gastrulation. Defective mesodermal folds are denoted by peach arrowheads in D and E. Abnormal serosal movements are denoted by a blue arrowhead in E.

Fig 2. Transverse views of control and Nv-dnmt1a pRNAi embryos during cellularization and at the onset of gastrulation.

Green, phalloidin (F-actin). white, DAPI (DNA). (A) Control embryo during mid cellularization. (B) Control embryo at the onset of gastrulation. Pink arrowheads demarcate the borders of the presumptive mesoderm. (B’) Zoomed in view of fully cellularized control embryo. Orange arrowhead points to the basal actin cable. (C) Nv-dnmt1a pRNAi embryo during mid-cellularization. White arrows point to examples of disordered nuclei. (D) Nv-dnmt1a pRNAi embryo at the onset of gastrulation. White arrows point to examples of non-cellularized, disordered nuclei. (D’) Zoomed in view of an Nv-dnmt1a pRNAi embryo that fails to fully cellularize. Orange arrowhead points to a non-cellularized nucleus (note the absence of the basal actin cable). (E) Table with n values of cellularized and non-cellularized control and Nv-dnmt1a pRNAi embryos. *** = p-value < 0.0001 determined by Fisher’s Exact Test. All scale bars (white lines in A-A”‘) represent 10 μm.

Fig 3. Global DNA methylation in control and Nv-dnmt1a pRNAi late blastoderm N. vitripennis embryos.

(A) An example of distribution of fractional DNA methylation in a control sample exhibiting the classical bimodal distribution. We defined methylated genes as genes with a minimum of 0.02 gene body methylation (shown as the dashed line) to classify methylated and unmethylated genes. (B) Violin plot demonstrating the reduction of whole genome DNA methylation in Nv-dnmt1a pRNAi samples compared to the control samples. We calculated the average fractional methylation of all methylated CpGs in the control samples and corresponding positions in the pRNAi samples (~151k methylated CpGs). (C) Distributions of fractional methylation of CpGs that are methylated at least one control sample demonstrate that CpGs lose DNA methylation in RNAi embryos. (D) The pattern of mean fractional methylation in the first and last four exons and introns of all methylated genes in control and pRNAi samples, divided into equal-sized bins. Control shown in red, pRNAi shown in blue.

Fig 4. Gene expression of methylated and unmethylated genes in control and Nv-dnmt1a pRNAi N. vitripennis embryos.

(A) Volcano plots showing differentially expressed genes between control and Nv-dnmt1a pRNAi in embryos of different developmental stages. X axis is log2 fold change in Nv-dnmt1a pRNAi samples relative to control samples. Y axis is log10 adjusted p-values. Golden dots are methylated, significantly differentially expressed (FDR-corrected p-value < 0.1). Purple dots are significant and DEGs that are unmethylated (FDR-corrected p-value < 0.1). Blue and grey are non-significant and represent methylated and unmethylated genes, respectively. (B) Distribution of methylated and unmethylated genes for all genes in the genome, differentially methylated genes (DMGs), down-regulated differentially expressed genes (Down DEGs), and up-regulated differentially expressed genes (Up DEGs). (C) The correlation between mean relative GBM change and mean relative expression change (calculated as log2-fold change) in the pRNAi samples compared to the control samples was positive and statistically significant (Spearman’s rank correlation coefficient = 0.37, p-value < 2.2e-16).

Fig 5. Violin plots of methylated and unmethylated differentially expressed genes (DEGs) in control and Nv-dnmt1a pRNAi samples.

(A) eggs, (B) early blastoderm embryos, (C) middle blastoderm embryos, and (D) late blastoderm embryos. p-values were determined by performing a 2-tailed T-test assuming unequal variance between RNAi and control. ns = not significant (P>0.05). * = P<0.05. *** = P <0.0001).