Genome binding/occupancy profiling by high throughput sequencing Methylation profiling by high throughput sequencing Other
Summary
Retrotransposons have invaded eukaryotic centromeres in cycles of repeat expansion and purging, but the function of centromeric retrotransposons, if any, has remained unclear. In Arabidopsis, centromeric ATHILA retrotransposons give rise to epigenetically activated short interfering RNAs (easiRNAs) in mutants in DECREASE IN DNA METHYLATION1 (DDM1), which promote histone H3 lysine-9 di-methylation (H3K9me2). Here, we show that mutants which lose both DDM1 and RNA dependent RNA polymerase (RdRP) have pleiotropic developmental defects and mis-segregation of chromosome 5 during mitosis. Fertility defects are epigenetically inherited with the centromeric region of chromosome 5, and can be rescued by directing artificial small RNAs to a single family of ATHILA5 retrotransposons specifically embedded within this centromeric region. easiRNAs and H3K9me2 promote pericentromeric condensation, chromosome cohesion and proper chromosome segregation in mitosis. Insertion of ATHILA silences transcription, while simultaneously making centromere function dependent on retrotransposon small RNAs, promoting the selfish survival and spread of centromeric retrotransposons. Parallels are made with the fission yeast S. pombe, where chromosome segregation depends on RNAi, and with humans, where chromosome segregation depends on both RNAi and HELLSDDM1.
Overall design
Whole genome bisulfite sequencing of ddm1 epigenetic recombinant lines in rdr1 rdr2 rdr6; Whole genome sequencing and whole genome bisulfite sequencing of rdr1 rdr2 rdr6 ddm1 suppressors; H3K9Me2 ChIP-seq in wild-type, rdr1 rdr2, rdr1 rdr2 rdr6, rdr1 rdr2 ddm1, rdr1 rdr2 rdr6 ddm1, rdr1 rdr2 rdr6 ddm1 ATHILA5-hp suppressor; CenH3 ChIP-seq in wild-type, rdr1 rdr2 rdr6, rdr1 rdr2 ddm1, rdr1 rdr2 rdr6 ddm1, rdr1 rdr2 rdr6 ddm1 ATHILA5-hp suppressor and rdr1 rdr3 rdr6 ddm1 episuppressor 3-75; ONT direct methylation sequencing of rdr1 rdr2 rdr6 ddm1 and rdr1 rdr2 rdr6 ddm1 ATHILA5-hp suppressor.
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