The essential role of Dnmt1 in gametogenesis in the large milkweed bug Oncopeltus fasciatus

Abstract

While DNA methylation is an important chromatin modification in many groups of organisms, the function of DNA methylation within the insects is unclear. The taxonomic distribution of DNA methyltransferase genes in insects is highly variable, as is the presence of methylated genomes. In the large milkweed bug, Oncopeltus fasciatus, we have shown the maintenance methyltransferase Dnmt1 is required for oocyte production but this appears to be unrelated to methylation given that demethylating somatic cells causes no loss of somatic cell function. One hypothesis is that Dnmt1 is affecting meiosis. Here we used RNAi to downregulate Dnmt1 in males at two stages where meiosis is occurring; during testis development and in adults replenishing sperm stores following sperm depletion. We found that downregulation of Dnmt1 in stages where meiosis is required resulted in the greatest disruption to spermatogenesis. Our results support the hypothesis that Dnmt1 is required for the transition of germ cells to gametes in O. fasciatus and that this function is conserved in male and female gametogenesis. In addition, the role of Dnmt1 was specific to the germ cells. Downregulation of Dnmt1 across all tissues resulted in a germline-specific phenotype. These results suggest that the reduction of methylation has a phenotype restricted to the germ cells. Our results raise the question of how a gene so critical in fitness across multiple insect species can have diverged widely across the insect tree of life.Significance StatementGiven the importance of DNA methylation in protection of the genome against transposable elements and transcriptional regulation in other taxonomic groups, the diversity in both levels and patterns of DNA methylation in the insects raises questions about its function and evolution. We show that the maintenance DNA methyltransferase, DNMT1, affects meiosis and is essential to fertility in milkweed bugs, Oncopeltus fasciatus, while DNA methylation is not required in somatic cells. Our results suggest that DNMT1 has a function independent of DNA methylation in germ cells. The evolutionary lability of a gene with such a fundamental fitness activity suggests that the function Dnmt1 in germ cell development is easily lost or replaced.

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

“…While our data strongly suggests that Dnmt1a regulates gene expression through GBM in Nasonia, there is evidence that DNMT1 orthologs have roles independent of their methylation functions in other insects. Dnmt1 knockdown data in milkweed bug Oncopeltus fasciatus and the beetle Tribolium castaneum suggest that Dnmt1 orthologs may have molecular functions unrelated to their methylase activity (29,52,53). We cannot exclude that a non-methylating function of Nv-Dnmt1a exists, and that it could contribute to the complex phenotypes we observed after knocking down Nv-Dnmt1a with pRNAi.…”

“…Results of Dnmt1 knockdown in the milkweed bug Oncopeltus fasciatus and the beetle Tribolium castaneum indicate that Dnmt proteins may have important functions not related to their methylation function 35,58,59 . In O.…”

“…However, no significant changes in gene expression were detected in ovaries, or in other adult tissues, raising the question of whether GBM is functionally relevant for oogenesis 35 . Subsequently, O. fasciatus Dnmt was shown to be crucial for meiosis in male testis, but again in and apparently GBM independent manner 58 .…”

DNA methylation is not a driver of gene expression reprogramming in young honey bee workers

“…For instance, even though the genome of the red flour beetle Tribolium castaneum is not methylated (60,61), the knockdown of a Dnmt1 ortholog resulted in offspring showing developmental arrest and high mortality (61). Similar developmental defects in response to a knockdown of Dnmt1 were observed in the reproductive system of females of the milkweed bug Oncopeltus fasciatus (37,62). These phenotypic alterations are not a consequence of transcriptomic changes, even though the Dnmt1 knockdown successfully reduced the global levels of DNA methylation (37).…”

“…S4). Therefore, the functional role of intragenic methylation in honey bees is likely to be associated with basal processes necessary for cell viability, e.g., cell division, genome integrity, and/or cell cycle progression (37,(61)(62)(63), rather than representing as a marker of tissue identity (Fig. 4), or acting as a dynamic sensor of changes in the social environment (Fig.…”