Piwi-piRNA complexes induce stepwise changes in nuclear architecture at target loci (2021)
Figure 2

Figure 2. Piwi-piRNA target TEs tethering to the nuclear periphery is Piwi-dependant.

(A) Scatterplot of DamID-seq signals (log2 ratio) for FlyBase genes (left) or genome integrated TEs (right) in EGFP-KD (control, x-axis) and Piwi-KD (y-axis) samples. Both x- and the y-axis is a log2 ratio. Gypsy signal with the greatest decrease upon Piwi-KD is indicated and shown in genome browser view in (B).

(B) DamID-seq signal densities are shown for OSCs with EGFP- and Piwi-KD at genomic regions harboring gypsy insertions with the greatest decrease upon Piwi-KD (indicated in (A)). Neighboring regions are annotated with protein-coding genes and LTR TEs. Note that this distribution of the reads includes multi-mapped reads.

(C) Boxplots showing fold changes in the expression of piRNA target or non-target TEs based on RNA-seq upon Piwi-KD (left), and the frequency of piRNAs, which target piRNA target TEs or non-piRNA target TEs (right). Definition of piRNA target and non-target TEs is in Fig EV1E. Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated using the Wilcoxon rank-sum test.

(D) Scatterplot as in (A) for EGFP-KD (control, x-axis) or Piwi-KD (y-axis) samples. Values were calculated for 90 TE consensus sequences (one value per one type of TE) (left). Scatterplot of RPM values for the same 90 TE consensus sequences in EGFP-KD (control, x-axis) or Piwi-KD (y-axis) samples examined by RNA-seq (right). The signals for mdg1, gypsy, and roo are labelled within the plots.

(E) Boxplot showing DamID-seq signals of piRNA target or non-target TEs upon EGFP (control)- or Piwi-KD. Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated using the Wilcoxon rank-sum test.

(F) Density plots for DamID-seq signals over consensus sequences from piRNA target (gypsy and mdg1, orange) and non-target TEs (roo, blue) in EGFP (control)- or Piwi-KD cells.

(G) Dot plot showing the correlation between the normalized density of indicated TEs per LAD (x-axis) and changes in Lam-Dam / Dam signals upon Piwi-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89).

(H) Oligo-FISH images for the representative piRNA target region described in Fig 4A, upon EGFP (control)- or Piwi-KD. Gray indicates DAPI staining for DNA, magenta indicates FISH signal, and green indicates Lamin immunofluorescence staining. Scale bar, 5μm. Ratio of distance from FISH signal to the nuclear periphery (DAPI surface) is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.

(I) Diagram illustrating relocation of piRNA-target TE coding chromosome regions away from the nuclear periphery.