DOI: 10.6084/m9.figshare.7559315.v1
title: MOESM1 of Dnmt1 is essential for egg production and embryo viability in the large milkweed bug, Oncopeltus fasciatus

description: Additional file 1: Fig. S1. Identification of O. fasciatus DNA methyltransferases. a Phylogenetic relationship of DNA methyltransferases identified the de novo (Dnmt3) and maintenance (Dnmt1) DNA methyltransferase in O. fasciatus. Node support with ≤ 0.5 posterior probability is indicated—other nodes are ≥ 0.95. Branch lengths are in amino acid substitutions per site. Species names are represented as abbreviations: Acy. pis.: Acyrthosiphon pisum, Aed. aeg.: Aedes aegypti, Aed. alb.: Aedes albopictus, Ano. gam.: Anopheles gambiae, Api. mel.: A. mellifera, Bom. mor.: Bo. mori, Cam. flo.: Camponotus floridanus, Cop. flo.: Copidosoma floridanum, Cul. qui.: Culex pipiens quinquefasciatus, Dro. mel.: Drosophila melanogaster, Har. sal.: Harpegnathos saltator, Mic. dem.: Microplitis demolitor, Nas. vit.: Nasonia vitripennis, Nic. ves.: Nicrophorus vespilloides, Onc. fas.: O. fasciatus, Cer. bir.: Ooceraea (Cerapachys) biroi, Pol. can.: Polistes canadensis, Pol. dom.: Polistes dominula, Sol. inv.: Solenopsis invicta, Tri. cas.: Tribolium castaneum, and Zoo. nev.: Z. nevadensis. b A to scale representation of Dnmt1 and protein domains identified in O. fasciatus and M. musculus. Fig. S2. Assessment of RNAi treatment targeting S-adenosyl-L-methionine (AdoMet) region (ds-dnmt1-2) of Dnmt1 produces similar results as ds-dnmt1-1. a Assessment of RNAi treatment targeting Dnmt1 using qRT-PCR demonstrates successful reduction in transcription in ovaries compared to control. b Whole ovaries from ds-dnmt1-2 females removed 12–14 days post-injection. c Control follicular epithelium nuclei. d ds-dnmt1-1 follicular epithelium nuclei. e ds-dnmt1-2 follicular epithelium nuclei. For c–e scale bar corresponds to 100 µm. Fig. S3. DNA methylation consequences following posttranscriptional knockdown of Dnmt1 are restricted to ovaries. a Assessment of RNAi treatment targeting Dnmt1 using qRT-PCR demonstrates successful reduction in transcription compared to control across all tissues sampled. Colored dots indicate independent biological replicates. b Genome-wide CG methylation level across tissues sampled. Numbers at the top of each bar correspond to a unique individual identifier. Fig. S4. Eggs laid in O. fasciatus ds-dnmt1-1 and control females. Number of eggs laid by ds-dnmt1-1 and control females 8 days post-injection. Dots indicate mean expression level, and error bars indicate standard error of the mean. Fig. S5. Expression of Dnmt3 is not affected by dsRNA targeting Dnmt1 (ds-dnmt1-1). Dots indicate mean expression level, and error bars indicate standard error of the mean. Fig. S6. mCG in A. mellifera and Z. nevadensis is not associated with transcription. a Gene expression level for deciles of increasing mCG (1–10) and unmethylated genes (UM). Error bars represent 95% confidence interval of the mean. b Regression of gene expression against a continuous measure of mCG among all genes with &gt; 0 FPKM and weighted mCG. Raw p values are provided for each regression, and significance (S) or non-significance (NS) is indicated in brackets following Bonferroni correction. c. The relationship between change in gene expression measured as the log2 fold-change of FPKM between A. mellifera dnmt3 knockdown and GFP control, and DNA methylation measured as the difference between A. mellifera dnmt3 knockdown and GFP control mCG levels. For gene expression, FPKM values were averaged across libraries. Density of bins corresponds to the number of genes with similar changes to expression and DNA methylation. Fig. S7. Expression changes of differentially methylated genes. A heatmap showing gene expression changes for genes that are differentially CG-methylated between O. fasciatus ds-dnmt1-1 and control. Expression was standardized by the highest value per gene per biological replicate to produce a relative fragments per kilobase of transcript per million (RFPKM) value. RFPKM were clustered using a hierarchical clustering method. Fig. S8. Loss of mCG in O. fasciatus ovaries has a limited effect on transcription. Combinational overlap of genes that are differential CG-methylated and expressed, and similarly CG-methylated and expressed between O. fasciatus ds-dnmt1-1 and control. Gene groups: differentially methylated gene (DMG), differentially expressed gene (DEG), similarly methylated gene (SMG)/unmethylated genes (UMG), and non-differentially expressed gene (non-DEG). More stringent thresholds were used to group genes as CG-methylated or unmethylated (see “Materials and methods”). Fig. S9. DNA methylation of TEs in O. fasciatus. Levels of mCG across the bodies and 1 kb flanking sequence of different annotated TEs in O. fasciatus. Table S1. PCR primers used to validate the presence of a single Dnmt1 ortholog in the O. fasciatus genome. Table S2. Primer sequences for use in producing template DNA for use in the MegaScript transcription kit to generate double-stranded RNAs for injection and for quantitative real-time PCR to assess expression levels of Dnmt1. Table S3. DNA methylation summary statistics for all species investigated in this study. Table S4. Output from edgeR v3.20.1 [69] with gene expression (fragments per kilobase of transcript per million mapped reads [FPKM]) for O. fasciatus ovaries ds-dnmt1-1 and control biological and technical replicates. Table S5. Gene expression (FPKM) for A. mellifera queen and drone brains. Table S6. Gene expression (FPKM) for A. mellifera dnmt3 knockdown and GFP control. Table S7. Gene expression (FPKM) for Z. nevadensis female worker at the final instar larva. Table S8. Output from edgeR v3.20.1 [69] with gene expression (FPKM) for O. fasciatus gut, head, and thorax ds-dnmt1-1 and control biological replicates. Table S9. Overlap between O. fasciatus (control) CG-methylated and unmethylated genes in none and ≥ 1 other insect species used in this study. Table S10. Output General Features File (GFF) from RepeatMasker v4.0.5 ( http://www.repeatmasker.org ). Table S11. Significance (p value) of TE expression between ds-dnmt1-1 and control tissues. Table S12. Gene Ontology (GO) terms for O. fasciatus v1.1 reference genome assembly [32] annotated genes. Table S13. Significantly enriched GO terms for the intersections between differentially methylated genes (DMG), similarly methylated genes (SMG), differentially expressed genes (DEG), and non-differentially expressed genes (non-DEG). Data S1. Unaligned and aligned DNA methyltransferase protein sequences in FASTA format, and a parenthetical phylogenetic tree in nexus format estimated from the aligned DNA methyltransferase protein sequences using BEAST v2.3.2 [51].