Retrotransposon addiction promotes centromere function via epigenetically activated small RNAs

Abstract

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 HELLS^DDM1.

Figure 1.. Fertility and floral defects of rdr1;2;6 ddm1 map to hypomethylated centromere 5.

a, Developmental defects of double, triple and quadruple mutants in RNA dependent RNA polymerase (rdr1, rdr2, rdr6) and DNA methylation (ddm1) in floral organ identity, leaf shape, and fertility (silique length). rdr1 rdr2 and rdr6 combinations are abbreviated as rdr1;2;6. b, Crossing scheme for constructing ddm1-derived epigenetic recombinant lines in an rdr1 rdr2 rdr6 background. Hypomethylated chromosomal regions derived from ddm1 mutants are inherited epigenetically in DDM1/DDM1 progeny, and are indicated in dark grey. c, Whole genome bisulfite sequencing of pooled fertile (pink) and sterile (blue) epigenetic recombinant lines (from b) indicates reduced methylation in the pericentromeric regions of chromosome 5.

Figure 2.. Epiallelic suppressors gain DNA methylation at the ATHILA5 retrotransposon in centromere 5.

a, Venn diagram of shared, hypermethylated differentially methylated regions (DMR) in 4 independent rdr1;2;6 ddm1 suppressors (2–69, 2–20, 3–72, 3–75), on chromosome 5. b, Boxplot analyses of DNA methylation levels in uniquely shared 1kb hyper-methylated DMR in each genotype. c, Uniquely shared DMR (black bar) corresponds to Cen5-ATHILA5 (blue LTRs) which is embedded in cen180 satellite repeats (purple box) and interrupted by ATHILA2. Genome Browser tracks display DNA methylation gains (blue) and losses (grey) in the 26kb region relative to rdr1;2;6 ddm1. d, Floral and chromosomal phenotypes of rdr1;2;6 ddm1 mutants are rescued by epiallelic suppressor 3–75. Mitotic chromosomes in root tip anaphase cells were stained with DAPI. A mis-segregating chromosome is indicated (white arrow).

Figure 3.. Cen5-ATHILA5 hairpin small RNA rescue rdr1;2;6 ddm1 phenotypes.

a, Cen5-ATHILA5 is embedded in sequenced (purple) and unsequenced (black) centromeric repeats on chromosome 5 (TAIR10 genome assembly). It encodes two ORFs (gray arrows), short regions of homology to mitochondrial DNA (light green) and a tRNA gene (orange). Synthetic hairpins hp1 through hp6 and probes used for northern analysis (Extended Data Fig. 5) are shown. Genome browser tracks display 21-nucleotide siRNA levels in indicated genotypes. b, RNAi hairpin hp5 strongly suppresses floral and fertility defects in rdr1;2;6 ddm1.

Figure 4.. Chromosome mis-segregation in rdr1;2;6 ddm1.

a, DNA FISH of root tip anaphase cells with Cy3-labeled DNA probes from chromosome 5 (red). Nuclei were counterstained with DAPI. Mis-segregating chromosomes are indicated with white arrows. b, Mis-segregation rate of chromosomes 1–5 in rdr1;2;6 ddm1 anaphase cells (n=100 abnormal cells) determined by FISH. c, Chromocenters were stained with DAPI, and quantified signals (n=30) illustrated by boxplots (right). 1, WT; 2, rdr1;2, 3 rdr1;2;6, 4, rdr1;2;6 ddm1, 5 rdr1;2;6 ddm1 hp5 RNAi.

Figure 5.. easiRNAs restore H3K9me2 and non-CG DNA methylation to ATHILA5 elements of chromosome 5.

a, Browser shot of DNA methylation in the different mutants, spanning the centromere of Chromosome 5 (Col-cen genome assembly). The dotplot reveals high identity between the cen180 repeats (light blue bar), with interspersed ATHILA elements (light green columns) and ATHILA5 elements (dark green columns). The CEN5-ATHILA5 used to design the RNAi hairpin is also shown (orange column, far right). DNA methylation was assessed by whole-genome bisulfite sequencing (WGBS), as well as by base calling from long-reads from Oxford Nanopore (ONT) using modbase2. mC calls are averaged in 5kb windows (y axis scale indicated for each context). b-c, Metaplots of (b) H3K9me2 and (c) DNA methylation (from ONT) over ATHILA5 (not including hp5 containing CEN5-ATHILA5) and all other ATHILA in the genome. Levels of H3K9me2 and non-CG methylation in the rdr1;2;6 ddm1 mutant are recovered specifically at ATHILA5 elements when the hp5 is present.

Figure 6.. Defective sister chromatid cohesion in rdr1;2;6 ddm1

a, Immunofluorescence for H3T3ph in root tip cells. Left panels exhibit mitotic prophase cells showing H3T3ph signals. Cells were counterstained with DAPI. In prophase cells, condensed DAPI dots are dispersed in the nucleus. H3T3ph dot sizes were calculated based on the nucleus size (Right panel). 100 dots from 20 nuclei were analyzed. b, DNA FISH in mitotic prophase cells with Cy5-labeled probes designed near the pericentromeric region of chromosome 5 (Left Panels). The right panel shows the number of Cy5 dots (1–4) in each nucleus. 50 nuclei were analyzed for each mutant.

Figure 7.. A model for the regulation of pericentromeric sister chromatid cohesion by DNA methylation and small RNAs.

The Arabidopsis pericentromere is maintained by DNA methylation and H3K9 methylation, and is essential for sister chromatid cohesion. When DNA methylation is lost, plants produce RDR6-dependent easiRNAs from ATHILA family retrotransposons, enriching H3K9 methylation at pericentromeric ATHILAs. Additional loss of easiRNAs causes impaired sister chromatid cohesion and severe mis-segregation of chromosome 5. The sterility, and the sister chromatid cohesion defect of chromosome 5 can be rescued by artificial small RNAs targeting retrotransposon ATHILA5, which re-establishes H3K9-methylated heterochromatin.