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. 2022 Oct 4;11:e80067. doi: 10.7554/eLife.80067

Figure 3. Rhino’s chromatin occupancy changes dramatically in CG2678/kipferl mutants.

(A) Western blot analysis verifying CG2678 frame shift (fs1) and locus deletion (Δ1) alleles using a monoclonal antibody against CG2678 (top; CG2678-PB is a minor protein isoform) and depicting Rhino levels in the absence of CG2678 (bottom). Ponceau staining: loading control. (B) Time-resolved hatching rates for eggs laid by w1118 control females in comparison to females carrying a CG2678 frame shift (fs1), locus deletion (Δ1), or tagged rescue construct instead of the CG2678 locus, respectively (AM, PM indicates egg laying time). Total number of eggs laid is indicated for each genotype. (C) Confocal images illustrating localization of GFP-Rhino in nurse cell nuclei of w1118, CG2678 locus deletion (Δ1), and CG2678,nxf3 double mutant females (scale bar: 5 µm). (D) Scatter plot of genomic 1-kb tiles contrasting average log2-fold Rhino ChIP-seq enrichment in ovaries with MTD-Gal4 driven CG2678/kipferl knock down versus control ovaries (average of two replicate experiments each). (E) Violin plots showing average log2-fold Rhino ChIP-seq enrichment in control (n=3) as well as CG2678/kipferl (n=2) or rhino (n=1) germline knockdown ovaries on Rhino-bound 1-kb tiles (defined in Figure 1D) in heterochromatin (HC) and chromosome arms (EC). piRNA clusters 38C, 42AB, and 80F are depicted separately. *** corresponds to P<0,001 based on student’s t-test. Box plots show median (center line), with interquartile range (box) and whiskers indicate 1.5x interquartile range. (F) UCSC browser tracks (ChIP-seq) depicting diverse Rhino domains in control and CG2678/kipferl germline knockdown ovaries (signal shown as coverage per million sequenced reads for one representative replicate). (G) Violin plots showing average log2-fold H3K9me3 ChIP-seq enrichment in control (n=3) and CG2678/kipferl (n=2) germline knockdown for Rhino-bound 1-kb tiles (defined in Figure 1D) in heterochromatin (HC) and along chromosome arms (EC). piRNA clusters 38C, 42AB, and 80F are depicted separately. *** and n.s. corresponds to p<0.001 or p>0.05, respectively, based on student’s t-test. Box plots show median (center line), with interquartile range (box) and whiskers indicate 1.5x interquartile range. (H, I) Jitter plots depicting the log2-fold Rhino ChIP-seq enrichments on transposon (H) and Satellite (I) consensus sequences in indicated genetic backgrounds. (J) Confocal images showing Rsp and 1.688 Satellite RNA FISH signal and GFP-Rhino in nurse cells of w1118 or CG2678/kipferl mutant flies (scale bar: 5 µm).

Figure 3.

Figure 3—figure supplement 1. CG2678/Kipferl impacts Rhino's nuclear localization and chromatin occupancy.

Figure 3—figure supplement 1.

(A) Western blots showing CG2678 or Rhino protein levels in ovarian lysate from indicated genotypes (Ponceau staining served as loading control). Changes in protein levels and isoform ratios between control and rhino depleted ovaries were not reproducible and are likely caused by differences in ovary morphology, which distort the protein composition across samples. (B) Confocal images showing immunofluorescence signal for CG2678 and Rhino in nurse cells of MTD-Gal4-driven control or CG2678 knockdown ovaries (scale bar: 5 µm). (C) Confocal images showing FLAG-tagged CG2678 and Rhino signal in nurse cells expressing an internally FLAG-tagged CG2678 in the CG2678 mutant background (see Figure 2B for location of FLAG tag; scale bar: 5 µm). (D) Confocal images showing the localization of GFP-Rhino with H3K9me3 (top), Deadlock (middle), or Nxf3 (bottom) in CG2678 null mutant nurse cells (scale bar: 5 µm). (E) Scatter plot of genomic 1-kb tiles contrasting average log2-fold enrichment for Rhino ChIP-seq signal in CG2678 knock out versus w1118 control ovaries (three and two replicate experiments, respectively). (F) Violin plots showing average log2-fold H3K9me2 ChIP-seq enrichment for Rhino-bound 1-kb tiles in MTD-Gal4 background in heterochromatin (HC) and along chromatin arms (EC) contrasting control (n=2) to RNAi-mediated CG2678/kipferl knockdown (n=1). Rhino-dependent piRNA clusters 38C, 42AB, and 80F are shown separately. Box plots show median (center line), with interquartile range (box) and whiskers indicate 1.5x interquartile range. (G) Confocal images showing the localization of piRNA cluster RNA via RNA FISH in respect to GFP-Rhino in nurse cells of w1118 or CG2678/kipferl locus deletion flies (scale bar: 5 µm). Imaging conditions were chosen to avoid photo bleaching and to optimally display the large Rhino-GFP accumulations to show that these are not corresponding to either of these clusters. (H) Confocal images showing Rsp and 1.688 Satellite RNA FISH signal in respect to GFP-Rhino in nurse cells of CG2678/kipferl,nxf3 double mutant flies (scale bar: 5 µm).