More than DNA methylation: does pleiotropy drive the complex pattern of evolution ofDnmt1?

Abstract

9DNA methylation is an important chromatin modification that can stably alter gene expression in 10 cells and maintain genome integrity in plants and vertebrates. The function of DNA methylation 11 outside of these well-studied systems, however, is unclear. Insects, in particular, represent an 12 understudied group. Variation in the level of DNA methylation and gains and losses in the 13 maintenance methyltransferase, DNMT1, across the insect tree of life suggests that there is much we 14don't understand about DMNT1 function and evolution. One constant across the studies examining 15 patterns of Dnmt1 expression in insects is that expression is consistently high in reproductive tissues 16 compared to somatic tissue. The explanation for this has been that DNMT1 is required in tissues that 17have high levels of cell division. Our previous study found that downregulation of Dnmt1 expression 18 in the milkweed bug Oncopeltus fasciatus results in the expected reduction of DNA methylation, no 19global changes in gene expression reflecting changes in DNA methylation, and the loss of the ability 20 to produce viable oocytes. Here, we show that females treated with ds-Dnmt1 RNA during larval 21 development have a more extreme phenotype; they lack oocytes entirely but develop a normal 22 somatic ovary. Our results indicate a specific role for DNMT1 in the formation of gametes and are 23 consistent with data from other systems, including Tribolium castaneum, a species does not have 24 DNA methylation. We propose that DNMT1 has multiple functional roles in addition to methylating 25 DNA, which explains its complex patterns of evolution, and suggests that previous inferences of 26 causation from associations are premature. 27

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

“…Thus, the phenotypic similarity between Dnmt1knockdown and Vasa knockdown testes in O. faciatus support the hypothesis that Dnmt1 may be required for the successful initiation and completion of meiosis in spermatocytes. More work remains to determine exactly what stage of sperm development is impacted by the decrease in Dnmt1 expression.The phenotype of knockdown of Dnmt1 is exacerbated when knockdown occurs prior to the stage of testis development when meiosis occurs, as was observed in female O. fasciatus(Amukamara et al 2020). One interpretation of this result is that Dnmt1 is required for successful progression through meiosis.…”

“…We also collected virgin males on the day of adult emergence and after sexual maturation at 7 days post-adult-emergence. We collected and tested these samples at the same time as the data reported for females (Amukamara et al 2020). Because 2nd and 3rd instar nymphs cannot be reliably sexed, the data presented here is the same as that presented in the previous publication.…”

“…One role emerging from knockdown and silencing studies across several insect species is that Dnmt1 is required for oogenesis (Schulz et al 2018, Bewick et al 2019, Gegner et al 2019, Amukamara et al 2020. For example, Dnmt1 is required for maintenance of DNA methylation following cell division in the milkweed bug, Oncopeltus fasciatus (Bewick et al 2019, Amukamara et al 2020, the downregulation of Dnmt1 using RNAi results in a reduction in methylation and also the cessation of oogenesis. However, it is unclear whether the effect on oogenesis is mediated by the reduction in DNA methylation (Amukamara et al 2020).…”

Debugging: Strategies and Considerations for Efficient RNAi-Mediated Control of the Whitefly Bemisia tabaci

“…Knocking out reproductive capacity is attractive because B. tabaci reproduction can be knocked out at several stages. Potential genes include those involved in gamete viability such as Boule , Vasa , or DNA methyltransferase 1 ( Dnmt1 ) [ 86 , 87 , 88 , 89 ]. The latter suggestion is a good example of the importance of understanding specific gene function.…”

“…However, methylation in insects does not appear to be universal, and some species such as the genetic model species D. melanogaster and Tribolium confusum lack Dmnt1 altogether [ 90 ]. Yet in Oncopeltus fasciatus , a hemipteran, co-authors have shown through RNAi knockdown that Dnmt1 has an essential function in gamete production in both males and females and that the silencing of Dnmt1 results in sterility without causing mortality or disrupting somatic tissue [ 86 , 87 ]. The Dnmt1 gene sequence has been identified in B. tabaci and successfully knocked down using RNAi [ 91 ].…”