Establishment of a forward genetic screening system to discover new proteins associated with DNA demethylation...

Active DNA demethylation mediated by Repressor of Silencing 1 (ROS1) plays a major role in counteracting gene silencing and spreading of DNA methylation. However, the molecular mechanisms behind the ROS1-mediated DNA demethylation pathway remain largely unclear. To gain a deeper understanding of this process, we established a novel HTC2-pUbq10-Ω-Kozak::LUC-1-based (H2UL-1-based) forward genetic screening system to identify more protein factors involved in such a pathway. H2UL-1 transgenic line was created by introducing a HTC2-pUbq10-Ω-Kozak::LUC construct into Col-0 plants, and the H2UL-1 seedlings emitted strong luminescence in the presence of luciferase's substrate luciferin; however, introduction of a ros1-4 mutation into the H2UL-1 background (designated H2UL-1/ros1-4 line) caused marked transcriptional silencing of Luciferase (LUC) gene. A terminator sequence downstream of LUC (named Ter^LUC) was highly methylated in H2UL-1/ros1-4 but not in H2UL-1 line, which may account for the silencing of LUC in the former. To test whether H2UL-1 line was suitable for identifying new candidate proteins, it was used as the starting material for mutant screening and subsequent gene cloning. Eventually, three genes were obtained, two of which were DMETER-Like 2 (DML2) and APEX1-LIKE (APE1L) (which are well-known for involvement in the ROS1-mediated DNA demethylation pathway) and one of which was Repressor of Gene Silencing 3 (RGS3), an SFMBT-like (SL) protein likely engaging in the modulation of chromatin conformation. Thus, the H2UL-1-based screening system appears to be effective in identifying candidate mutants (including new ones) and have the capacity to recover novel protein factors involved in ROS1-mediated DNA demethylation.