Piwi-piRNA complexes induce stepwise changes in nuclear architecture at target loci (2021)
Figure 5

Figure 5. Nxf2 regulates chromatin localization and decreases of intra-TAD interaction.

(A) Immunoprecipitation (IP) from OSC lysate using anti-Lam, or myc antibody, followed by WB using indicated antibodies. Mouse immunoglobulin G (n.i.) was used for control IP. Note that OSCs used at the right panel is a line which stably expresses myc-Nxf2.

(B) DamID-seq signal densities are shown for OSCs with EGFP (control)- and Nxf2-KD at genomic regions harboring gypsy insertions with the greatest decrease upon Nxf2-KD (locus shown in Fig 2B). Neighboring regions are annotated with protein-coding genes and LTR TEs. Note that this distribution of the reads includes multi-mapped reads.

(C) Scatterplot of DamID-seq signals (log2 ratio) for EGFP (control)-KD (x-axis) or Nxf2-KD (y-axis) samples. Values were calculated for 90 TE consensus sequences (one value per one type of TE) (left). Scatterplot of RPKM 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.

(D) Boxplot showing Lamin DamID-seq signals of piRNA target or non-target TEs upon EGFP (control)- or Nxf2-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.

(E) 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 Nxf2-KD cells.

(F) Dot plot showing the correlation between the normalized density of indicated TEs per LAD (x-axis) and changes in Lam-Dam / Dam signals upon Nxf2-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89).

(G) Differential interaction heatmap for chromosome 3L (40k resolution) in log2 fold change, showing increased (red) and decreased (blue) interacting bins upon Nxf2-KD.

(H) Differential intra-TAD interaction density score upon Nxf2-KD (upper), centering TE insertions. Heatmap showing changes in the intra-TAD interaction score of each piRNA target TE insertion sites (lower). The results are shown for piRNA target TE and non-piRNA target TE insertions.

(I) Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in intra-TAD interactions upon Nxf2-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89).

(J) Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in inter-TAD interactions upon Nxf2-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89).

(K) Dot plot showing the correlation between the change of coverage of LAD for each TAD (x-axis), and changes in intra-TAD interactions upon Nxf2-KD (y-axis). The trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89).

(L) Oligo-FISH images for the reporter of the tethering system, at 0h (control), 48h hours, 96 hours after transfection of λN-Nxf2 expression vector. Gray indicates DAPI staining for DNA, magenta indicates FISH signal, and green indicates Lamin staining. Scale bar, 5μm. The 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.