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

Figure 2. The ZAD-zinc finger CG2678 interacts with Rhino and binds Rhino domains genome-wide.

(A) Genomic CG2678 locus depicting the two annotated transcripts and CG2678 protein domain architecture (location of the frame shift mutation (red asterisk), internal 3xFLAG affinity tag (red circle), and cleavage sites for full locus deletion (red arrows) are indicated). (B) Volcano plot showing fold enrichment of proteins determined by quantitative mass spectrometry (Doblmann et al., 2019) in GFP-Rhino TurboID samples versus nuclear GFP TurboID control (n = 3 biological replicates; statistical significance based on two-sided t-test; P values corrected for multiple testing (Benjamini–Hochberg), see also Figure 2—figure supplement 2A and Figure 2—source data 1). (C, D) Confocal images of nurse cell nuclei (C) and germarium (D). Single channel and merged color images depict immunofluorescence signal for endogenous CG2678 (left, cyan) and Rhino (middle, magenta). Scale bar: 5 µm, dotted line: nuclear outline based on DAPI. (E) Scatter plot depicting correlation of log2-fold Rhino versus CG2678 ChIP-seq enrichment in w1118 ovaries (average of 2 replicates each). CG2678-only tiles are highlighted in green and were defined by significantly higher enrichment for Rhino than CG2678 (n=2, Z-score=3), plus a Rhino enrichment of max. 4-fold in two independent experimental replicates. (F, G) UCSC browser tracks illustrating CG2678 signal at diverse Rhino domains (F) and at CG2678-only peaks (G). ChIP-seq signal is shown as coverage per million sequenced reads for w1118 ovaries for one representative replicate. (H) Violin plots depicting log2-fold enrichment of H3K9me2 (orange, n=1) and H3K9me3 (brown, n=2) at euchromatic 1-kb tiles bound by neither Rhino nor CG2678, both proteins, or CG2678 only. Classification into groups was performed based on binary cutoffs for Rhino (fourfold) and a linear fit for CG2678 co-occupancy in two independent replicate ChIP-seq experiments from w1118 ovaries to extract CG2678-only tiles highlighted in (E).

Figure 2—source data 1. Imputed enrichment and p-values (n=3) for quantitative mass spectrometry data underlying Figure 2B.

Figure 2.

Figure 2—figure supplement 1. Multiple sequence alignment of selected reciprocal best CG2678 homologs generated with mafft v7.505 (Rozewicki et al., 2016) and visualized with espript v3.0 (Robert and Gouet, 2014).

Figure 2—figure supplement 1.

Protein sequences included in the alignment were obtained from the NCBI non-redundant database, their protein accessions being XP_033162862, XP_032579476, EDX12039, XP_039231961, XP_039493628, XP_043654173, XP_044252272, XP_017119505, XP_017057177.
Figure 2—figure supplement 2. CG2678 is expressed in ovaries but not in testes and binds Rhino domains on chromatin.

Figure 2—figure supplement 2.

(A) Cartoon illustrating flexible GFP-nanobody-TurboID setup used for the characterization of proteins in close proximity to GFP-tagged Rhino. (B) Bar graph indicating the expression pattern of CG2678 (Flybase modENCODE Anatomy RNA-Seq; L: larva; P: pupa; A: adult; M: male; F: female). (C) Confocal image of anterior tip of a testes stained for endogenous CG2678 (left, cyan) and Rhino (middle, magenta) with enlarged view of one nucleus (dotted line: nuclear outline based on DAPI; scale bar: 5 µm). (D, E) Scatter plots depicting the correlation of log2-fold Rhino versus CG2678 ChIP-seq enrichments in MTD-Gal4 (D; n=2) or iso1 ovaries (E; n=1).