Knockdown of Dnmt 1 links Gene body DNA methylation to 6 regulation of gene expression and maternal-zygotic transition in 7 the wasp Nasonia 8 9 10

23 Gene body methylation (GBM) is an ancestral form of DNA methylation 24 whose role in development has remained unclear. Unlike vertebrates, DNA 25 methylation is found exclusively in gene bodies in the wasp Nasonia vitripennis, 26 which provides a unique opportunity to interpret the role of GBM in development. 27 We confirmed that parental RNAi (pRNAi) knockdown of a DNMT1 ortholog (Nv28 Dnmt1a) in Nasonia leads to embryonic lethality and failures in cellularization and 29 morphogenesis. Using whole-genome bisulfite sequencing, we found a 30 widespread loss of GBM in Nv-Dnmt1a pRNAi embryos. Using RNAseq, we 31 found that methylated genes that lost GBM in the pRNAi samples were 32 exclusively downregulated during zygotic genome activation. Unexpectedly, 33 nearly all affected unmethylated genes were up-regulated after pRNAi. Lack of 34 proper clearance of mRNAs and abnormal activation drive this up-regulation, 35 indicating critical roles for Nv-Dnmt1a and GBM in the maternal-zygotic transition 36 (MZT) in the wasp, despite their absence in Drosophila. 37 38 Background 39 The maternal-zygotic transition (MZT) is an essential stage in multicellular 40 eukaryotic development and represents the transition of developmental control 41 from maternal genome products provided to the egg during oogenesis to the 42 products of newly formed zygotic genome 1, . A major hallmark of the handover 43 to zygotic control is the coordination of the highly regulated decay of maternal 44 mRNAs with the activation of transcription from the zygotic genome. 45 The interactions between the maternal and zygotic genome have been 46 most comprehensively demonstrated in the insect model Drosophila 47 melanogaster. Maternal transcript destabilization is carried out in part by 48 maternally deposited mRNAs encoding RNA binding proteins (RBPs) such as 49 smaug and brain tumor (brat) 3, . These factors are aided by the transcription 50 factor Zelda (Zld), which promotes miRNA expression that further destabilize 51 maternal transcripts. Under normal circumstances Zld also increases chromatin 52 accessibility and helps activate thousands of zygotic genes to their proper level 5, 53 6, . However, if clearance of maternal transcripts is impeded, the zygotic genome 54 is not properly activated. Thus, disruption of either zygotic genome activation, or 55 maternal transcript clearance leads to embryonic lethality. In both cases death is 56 due to failures in the earliest developmental events that are dependent on the 57 zygotic genome: cellularization of the syncytial blastoderm, and gastrulation 5, 8 . 58 Most of the strategies for regulating the D. melanogaster ZGA (e.g., 59 histone modification, chromatin packaging, action of miRNAs, mRNA 60 degradation), and many of the molecules involved, are conserved throughout the 61 animals . However, D. melanogaster lacks a major class of genomic regulation, 62 in the form of methylation of cytosines in CpG dinucleotides (CpG methylation) 10, 63 . CpG methylation is crucial for regulating gene expression in most complex 64 eukaryotes 12, , and has a significant role in regulating the MZT in many species 65 14, . Thus, its absence in D. melanogaster, and in the other major invertebrate 66 model system C. elegans, makes it difficult to elucidate the molecular 67 mechanisms of DNA methylation in early development. 68 CpG methylation is catalyzed by DNA methyltransferases 1 and 3 69 (DNMT1 and DNMT3), which are conserved and ancient components of the 70 eukaryotic genomic toolkit , but have been lost in Dipterans, including D. 71 melanogaster 17, . However, both DNMT1 and 3 orthologs are found in many 72 other insect taxa including hemimetabolous insects 19 and within the 73 holometabolous lineage Hymenoptera 20, 21, 22, . 74 The most well understood functional impact of CpG methylation, 75 elucidated from studies of mammalian model system, is in silencing gene 76 expression via methylation of cis-regulatory sequences, particularly of normally 77 unmethylated clusters of CpGs (CpG islands) 13, . However it is becoming clear 78 that cis-regulatory regulation at CpG islands is not a ubiquitous mechanism . A 79 more universal, and likely ancestral 20, , form of DNA methylation occurs 80 between the transcription start site and transcription end site of a gene, and is 81 known as gene body methylation (GBM) . 82 Previous studies have correlated GBM with stable and higher levels of 83 gene expression than genes without GBM 26, 28, 29, 30, , but its role in regulating 84 gene expression in specific developmental contexts such as the MZT is poorly 85 understood. Whole-genome bisulfite sequencing (WGBS) studies have shown 86 that DNA methylation patterns are dynamic during the MZT in vertebrate model 87 systems 32, 33, . However, such methylation studies in vertebrates have focused 88 on cis-regulatory regions, including CpG islands in promoters and intergenic 89 regions. Disruption of methylation at CpG islands often has profound phenotypic 90 effects, while the role of GBM is less well understood. Because manipulating 91 DNA methylation machinery in vertebrate models affects both CpG islands and 92 GBM, it is difficult to identify functions of GBM that are independent of CpG 93 island methylation. 94 In contrast to vertebrates, DNA methylation occurs almost exclusively at 95 gene bodies in insects that have retained DNA methyltransferases 1 and 3 96 (DNMT1 and DNMT3) 19, 22, 35, . Thus, such insect model systems have the 97 potential to make significant contributions in understanding the role of GBM in 98 development. A particularly attractive system with a full methylation toolkit and 99 exclusively gene body methylation is the wasp Nasonia vitripennis 36, 37, . 100 There are three DNMT1 paralogs in the N. vitripennis genome, and one of 101 them, Nv-DNA methyltransferase 1a (Nv-Dnmt1a), was shown to be essential for 102 embryogenesis (the other two showed no obvious phenotype). Therefore, N. 103 vitripennis offers an exciting opportunity to functionally study the specific role of 104 gene body methylation in a well-defined developmental system. In this work, we 105 set out to experimentally investigate the role of Nv-Dnmt1a and thus identify a 106 role for gene body methylation in early embryogenesis of N. vitripennis. 107 Our detailed analysis of the developmental effects of Nv-Dnmt1a revealed 108 major defects in cellularization and morphogenesis of the early embryo, which 109 are phenotypes typically seen after disruption of the MZT in insects. Using 110 parental RNAi (pRNAi) and WGBS we found that the vast majority of gene body 111 methylation is lost when Nv-Dnmt1a is knocked down. We then show that loss of 112 gene body methylation during zygotic genome activation is correlated with 113 reduced gene expression from the affected locus during zygotic genome 114 activation. Furthermore, we found that genes that are not methylated, but whose 115 expression changed after Nv-Dnmt1a knockdown, were primarily upregulated in 116 knockdown embryos relative to control. Nv-Dnmt1a knockdown profoundly 117 altered the levels of expression of thousands of genes in the early embryo. 118 119 Results 120 Nv-Dnmt1a function is required for early embryonic events that require 121 proper regulation of the zygotic genome 122 The initial characterization of Nv-Dnmt1a showed that knockdown through 123 parental RNAi (pRNAi), where female embryos are injected with dsRNA , led to 124 lethality of embryos produced by injected females . Understanding the precise 125 developmental failure(s) that lead to embryonic lethality should provide insight 126 into the functional importance of Nv-Dnmt1a for early embryonic development. 127 Therefore, we examined the embryonic development following Nv-Dnmt1a 128 pRNAi in more detail. 129 We first characterized the events of the first half of embryogenesis (0-15 130 hours, from egg lay to the completion of gastrulation) using time lapse imaging of 131 Nv-Dnmt1a pRNAi embryos. Similar to D. melanogaster, early development is 132 very rapid in N. vitripennis. During early embryogenesis, nuclei divide rapidly, 133 simultaneously, and without the formation of cell membranes to fill the large, 134 unicellular egg with syncytial nuclei. After 7 mitotic division cycles, nuclei migrate 135 to the cortex of the embryo, forming a syncytial blastoderm , consisting of a 136 single layer of nuclei populating the entire cortex of the egg. We observed no 137 obvious differences between control embryos and Nv-Dnmt1a pRNAi embryos 138 during the first 11 syncytial divisions. Syncytial blastoderm formation was 139 normal, and the last 4 divisions showed the characteristic increase in length 140 relative to earlier, pre blastoderm, cycles (Figure 1A). 141 The first obvious defect occurred at the 12 division cycle (Figure 1B, 1D). 142 There is normally a significant pause at this stage, as the nuclei are surrounded 143 by plasma membrane and thus transition from a syncytial to a cellular state, a 144 process referred to as cellularization (Figure 1A). Normally, cellularization 145 directly precedes the first morphogenetic movements of the embryo (gastrulation 146 and migration of the extraembryonic serosa) . In this work, control embryos 147 began gastrulation 193 minutes after the onset of nuclear cycle 12, on average 148 (n=5, SD = 13.12, (Figure 1A)), similar to our previously published results in 149 untreated embryos . Apparent morphogenetic movements in Nv-Dnmt1a pRNAi 150 embryos began significantly earlier, on average, 171 minutes (n=5, SD = 5.98) 151 into cycle 12 (Figure 1A). 152 In addition to being premature, these movements were abnormal. In Nv153 Dnmt1a pRNAi embryos, a line of cells at the anterior end of the embryo began 154 to ingress posteriorly (Figure 1D) which was not seen in control embryos (Figure 155 1B). In addition, internalization of the mesoderm and migration of the serosa from 156 the dor