Genetic screening of crossover rate mutants using activation tagging

Abstract

Meiosis is the specialized cell division that halves the number of chromosomes from parents and contributes to genetic diversity. During meiosis, homologous chromosomes pair and undergo chromosomal exchanges, called crossovers. Meiotic recombination initiates with the programmed DSB formation by SPO11. Numerous DBSs are repaired using homologous DNA templates of sister chromatids or non-sister chromatids but only a subset of them are repaired to crossovers while the remaining DSBs are repaired using sister chromatids or repaired to non-crossovers (gene conversions). Generally, crossover number tends to be 1-3 per chromosome pair in plants, indicating that crossovers are strongly limited. Two pathways to crossover are conserved: Class I and Class II pathways. Class I crossovers depend on a group of pro-crossover factors, called ZMM proteins, representing 85-90% of crossovers. Class I crossovers are sensitive to crossover interference that inhibits closely spacing of crossovers along chromosomes. By contrast, Class II crossovers are non-interfering and rely on structure specific endonuclease MUS81. Three groups of anti-recombination factors such as FANCM, FIGL1, and RECQ4A that restrict Class II crossovers and promote non-crossovers have been identified by a suppressor screening of Class I crossover zmm mutants in Arabidopsis. By contrast, HIGH CROSSOVER RATE1 (HCR1), HCR2, and HCR3 that act in class I pathway have been identified through a genetic screening using a fluorescent seed-based crossover rate measurement method and EMS mutagenesis. To identify new pro-crossover factors, I have performed a genetic screen for high crossover rate dominant (hcr-D) mutants through activation-tagging mutagenesis and fluorescent seed-based crossover reporter. I have isolated eight hcr-D mutants. Using Nanopore sequencing I have mapped locations of T-DNA containing enhancers in hcr-D mutants (hcr10-D to hcr16-D). By RT-qPCR analysis I confirmed that candidate genes close to the T-DNA insertion were overexpressed in hcr-D mutants. Among 8 hcr-D mutants, I have further characterized hcr11-D. I found that VIP3/SKI8 is overexpressed in hcr11-D. In yeast, VIP3 homolog, SKI8 is known to interact with SPO11 as a key component of DSB machinery. However, DSB formation was unchanged in Arabidopsis vip3/ski8. VIP3/SKI8 is required to activate gene transcription as a component of PAF1 complex. Consistently, I have observed that gene expression of HEI10 encoding an E3 ligase is increased in hcr11-D and decreased in vip3 mutant, indicating that VIP3 is required for transcriptional activation of HEI10, the key dosage-dependent ZMM pro-crossover factor. I confirmed that crossover rate is increased in the primary and secondary transgenic lines (T1, T2) that express VIP3 using a meiotic specific gene DMC1 promoter. In addition, genotyping-by-sequencing was performed using about 96 hcr11-D Col/Ler F2 plants from self-fertilized hcr11-D Col/Ler F1 plants to generate a genome-wide crossover map. I observed that the number of crossovers is increased in the genome scale, with substantial increase in chromosome 4 and 5. Using yeast two-hybrid (Y2H) assay I found that VIP3 interacts with MTOPVIB and DFO, components of plant DSB machinery, suggesting the possibility that VIP3 may contribute to DSB formation when overexpressed. Together, I found that VIP3 is required for HEI10 transcription, and characterization of activation-tagging hcr-D mutants will lead to discover novel dosage-dependent pro-crossover factors.