Figure 1.
Chromatin organization in piRNA mutant embryos.
A. Immunostaining for α-tubulin (green) and DNA (blue) in 0–30-min-old embryos showing chromatin fragmentation and chromatin fusions in aub and armi mutant embryos during meiosis II. Scale bar is 15 µM. B. Cross-section of 0–3-hr-old embryos during syncytial mitotic divisions showing DNA fragmentation and chromatin bridges during segregation in aub and armi mutants. Scale bar is 10 µM. C, D. Dual-label FISH for two Y-chromosome-specific satellites, (AATAC)n in green and (AATAAAC)n in red, with DNA in blue showing mis-segregation of these repeats in aub and armi embryos (C). In contrast, embryos undergoing cleavage mitotic divisions show both the labels in most of the segregating chromatids in aub (D).
Figure 2.
ligIV–dependent telomere fusions in piRNA mutants.
A. Two-color FISH for a pair of daughter nuclei in anaphase, labeled for centromeric dodeca satellite (green) and telomeric transposon, HeT-A (red) with DNA (blue) showing telomeres are fused in piRNA mutants. B. Immunostaining for microtubules (green) and DNA (blue) in 0–3 hr-old embryos showing suppression of chromatin bridge formation in ligIV;aub embryos. Scale bar is 10 µM. C. Ratio of anaphase/telophase bridges to total anaphase/telophase figures in different genotypes. The data for multiple samples were compared using Anova test, and sample mean was plotted with standard error of mean (SEM) as error bars. A two-tailed t-test was performed for certain pairs and p-values are noted on the graph.
Figure 3.
Mutations in aub and armi disrupt assembly of the telomere protection complex.
A. Schematic showing transposon arrays at Drosophila telomeres. The HeT-A transposon 3′ and 5′-UTRs are in red and yellow respectively, and the ORF is in blue. B, C. Binding of the telomere protection complex proteins HOAP and HP1 to HeT-A. Chromatin Immunoprecipitation (ChIP) was used to recover bound DNA, and the percent of input chromatin precipitated was determined by qPCR. Fold change in binding relative to wild type is shown, and was calculated by dividing mutant by wild type (wt) values. D. Genomic copy number for HeT-A and TART. Copy number was determined by qPCR, using the single copy Rp49 gene as an internal standard. Gaiano is a wild-type stock previously shown to carry additional telomeric transposon repeats.
Figure 4.
piRNAs linked to a 4th chromosome cluster containing telomeric transposon fragments.
A. Length histograms showing plus genomic strand (blue) and minus genomic strand (red) mapping piRNAs in wt, armi, aub, rhi and ago3 mutants. The relative abundance is normalized to sequencing depth and is plotted on the y-axis. Note that sense strand of the transposon fragments in this cluster are on the minus genomic strand, and that the scales differ. Preferential loss of shorter piRNAs from aub and armi leads to a prominent endo-siRNA peak at 21 nt (marked by a black arrow). B. Abundance of longer (23–29 nt) plus strand (blue) and minus strand (red) piRNAs in the indicated mutants relative to their respective wild-type controls. All four mutations reduce plus strand piRNAs, which are anti-sense to the telomeric transposons. C. 19–22 nt genomic plus and minus strand piRNAs in the indicated mutants. All four mutations reduce plus strand RNAs. However, minus strand species are retained at near wild type levels in both rhi and ago3 mutants. For panels B and C, bars show normalized reads in mutants divided by normalized reads in wild-type controls.