Extended Data Figure 4: Moonshiner mutants reveal highly specific function at Rhino-bound piRNA clusters.
From: A heterochromatin-dependent transcription machinery drives piRNA expression
a, Schematic of the moonshiner frameshift alleles generated by CRISPR/Cas9. b, piRNA levels from ovaries with indicated genotype (relative to wild type) mapping uniquely to indicated piRNA clusters. c, Left: deregulation of steady-state transposon transcript levels (RNA-seq; sense only) in ovaries of the indicated mutants fly strains. Right: changes in corresponding piRNA levels (antisense only). The y axis values show log2(fold change) of transcripts per million values relative to wild type. Each bar represents one transposon consensus sequence (n = 73; shown are only transposons with minimum expression of RNA-seq transcripts per million > 5 in any library). Sorting of transposons in all panels is identical. The plotted values are available as figure source data. d, Rhino occupancy at indicated major piRNA clusters as well as all other Rhino-bound loci is shown as boxplot quantification (n = 1-kb windows analysed for each group) of Rhino ChIP-seq read coverage in the indicated genotypes. Boxplots are defined as in Fig. 3c; ***P < 0.0001 based on Mann–Whitney–Wilcoxon non-parametric tests. e, Genome browser panel showing read coverage at cluster80F of the data underlying the log2(fold change) tracks shown in Fig. 3b. Shown are RNA-seq (green), Pol II ChIP-seq (red), and ChIP-seq input samples (purple) generated from the indicated genotypes. f, RNA-seq transcripts per million values for canonical genes compared between control and moonshiner−/− (left) or rhino−/− (right); key genes related to Moonshiner biology are highlighted in orange. Abbreviation rSpearman denotes the Spearman correlation coefficient for each data set pair. g, Representative confocal images underlying the quantitative RNA FISH-based detection of piRNA precursors from cluster20A (Rhino-independent) and cluster42AB (Rhino-dependent) in germline nuclei of wild-type and moonshiner mutant ovaries. h, Example confocal images of germline nuclei stained for of DNA (DAPI) and nuclear pore complexes (wheat germ agglutinin, WGA-488), which were used to define the nuclear region in whole-nucleus Z-stack images acquired in parallel with images of RNA FISH signal. i, Example single-plane images of dual-channel RNA FISH quantification of whole-germline nuclei. RNA FISH signal within the nuclear regions (left, segmented using DAPI and WGA-488 signal) was used to define regions of interest (right), representing active sites of piRNA cluster transcription6. Signal in the foci was subsequently quantified for whole nuclei.
