Functional modules of DEMETER 5-methylcytosine DNA glycosylase necessary for seed development in Arabidopsis

Abstract

DNA methylation is a key epigenetic mark that regulates gene expression. In Arabidopsis, DEMETER (DME) DNA glycosylase specifically removes 5-methylcytosine (5mC) from DNA and induces gene imprinting in endosperm. In heterozygous dme mutants, 50% of seeds are aborted due to a maternal parent-of-origin effect. There are three other DME-like genes such as ROS1, DML2, and DML3 in the Arabidopsis genome, and all DME family proteins share the similar domain structures. Besides the central DNA glycosylase domain, two additional domains (domains A and B) exist and all these are required to excise 5mC. These three domains are interspersed with the interdomain regions (IDRs) which are highly variable in sequence and length among the family members. To identify the minimal fragment necessary for 5mC excision, the DMEΔ(149-677)ΔIDR1::lnk, where the domains are cut and pasted, were engineered. The modified DME fragment still retains biochemical activity. This indicates that the three domains are enough for DME function in vitro. In the first chapter, DMEΔ(149-677) and DMEΔ(149-677)ΔIDR1::lnk which have DNA glycosylase activity were expressed in the central cell using DME promoter. The modified DME can rescue seed abortion in dme mutants. This demonstrates that the three conserved domains are functional modules which are necessary and sufficient for both in vivo and in vitro functions of DME. In addition, DMEΔ(149-677) could maintain the DME function stably in the next generation. Homozygous dme mutants with lethal phenotype were recovered and abnormal seed and flower phenotype were observed in T2 DMEΔ(149-677)ΔIDR1::lnk population. The results implies that DMEΔ(149-677)ΔIDR1::lnk might be able to have hyperactivity function of DME and that DNA methylation and floral morphology are related in Arabidopsis. In the second chapter, a novel DNA methylome analysis tool using DME was proposed. After DME DNA glycosylase reaction in which DME recognizes and removes mC in Arabidopsis genome, radioactively labeled cytosines were refilled in the vacant position by BER pathway. Here, dot-blot analysis was conducted and DNA methylation pattern was visualized for the specific regions of genome. The result suggests that DME treatment differentiates between hyper- and hypo-methylated genome fragments. If the method would be applied to fluorescent labeling system and tiling array, DME-chip could become the easy and fast DNA methlyome analysis tool through the single tube DME reaction.