Structure prediction and phylogenetic analyses point to a Rhino-specific residue involved in binding Kipferl.

(A) Domain organization of Kipferl and Rhino, with the AlphaFold pLDDT score plotted as a measure of order or disorder alongside. Red boxes indicate the smallest interacting fragments identified by yeast two-hybrid experiments by Baumgartner et al. (Baumgartner et al., 2022). ZAD, Zinc finger associated domain; ZnF, Zinc finger; CD, chromodomain; CSD, chromo shadow domain (B) Multiple sequence alignment of HP1 family proteins in five selected species harboring an unequivocally identified Kipferl homolog (see Figure 1 – figure supplement 3). Rhino-specific amino acid residues are indicated. Protein accessions and identifiers are documented in Supplementary File 1. Multi-Relief representation indicates residues that differ significantly in Rhino homologs versus other HP1 variant proteins. (C) PAE plot for the top ranked AlphaFold2 Multimer prediction of the Rhino chromodomain with the Kipferl ZnF cluster 1 (left) and structure of the complex in cartoon representation (Rhino, blue; Kipferl, green), together with the H3K9me3 peptide (orange) as observed in a Rhino – H3 crystal structure (PDB ID 4U68). Key residues of Rhino’s aromatic cage and H3K9me3, as well as of Kipferl’s C2H2 ZnF4 are shown in sticks representation. Only the interacting ZnF4 is shown. Depicted in the inset are Rhino G31 and HP1 D31, with HP1 (PDB ID 6MHA) superimposed on Rhino chromodomain residues 26-57 (RMSD = 0.55 Å), together with Kipferl V285 and F286, illustrating that D31 would lead to steric clashes with Kipferl.

G31 point mutations do not affect Rhino’s ability to bind H3K9me3.

(A) Line graph summarizing SEC-MALS results for the examined Rhino chromodomain constructs. The in solution molecular weight is indicated for each construct. (B) Isothermal titration calorimetry results showing the binding of indicated Rhino chromodomain constructs to the H3K9me3-modified histone tail peptide.

The rhinoG31Dpoint mutation recapitulates the mutant phenotypes observed for Rhino and Kipferl in each other’s null mutant background.

(A) Bar graph depicting female fertility as egg hatching rate in percent of laid eggs for indicated genotypes. (B) Confocal images showing immunofluorescence signal for Kipferl and Rhino in egg chambers of indicated genotypes. Zoomed images display one representative nurse cell nucleus (labeled by white asterisk in panel A) per genotype (scale bar: 20 µm).

The RhinoG31D point mutation uncouples Rhino and Kipferl at chromatin.

(A) UCSC genome browser screenshots depicting the ChIP-seq signal for Rhino and Kipferl at diverse Rhino domains in ovaries of the indicated genotypes (signal shown as coverage per million sequenced reads for one representative replicate). (B-E) Scatter plot of genomic 1-kb tiles contrasting average log2-fold ChIP-seq enrichment for Rhino (B-D) or Kipferl (E) in ovaries of the indicated genotypes (values displayed represent the average of two to three replicate experiments)

Kipferl-independent functions of Rhino are not affected by the G31D point mutation.

(A) Confocal images showing Rsp and 1.688 g/cm3 Satellite RNA FISH signal in nurse cells of indicated genotypes (scale bar: 5 µm). (B, C) Jitter plots depicting the log2-fold enrichments for Rhino ChIP-seq on consensus sequences of Satellites (B) or Rhino-dependent transposons (C) in ovaries with indicated genetic backgrounds. (D, G) Jitter plots depicting the length-normalized antisense piRNA counts on Satellite consensus sequences derived from ovaries (D) or testes (G) of indicated genetic backgrounds. (E, F) Box plots depicting the log2 fold change of piRNA counts (compared to w1118 control) per 1kb tile for major piRNA clusters in ovaries (E) or testes (F) of the indicated genotypes. The number of tiles per piRNA cluster is indicated as n.

Multiple sequence alignment of HP1 family proteins across Drosophila species. Further details on protein accessions and identifiers are documented in Supplementary File 1. Multi-Relief representation indicates residues that differ significantly in Rhino homologs versus other HP1 variant proteins.

Phylogenetic tree illustrating the evolutionary relationship of zinc finger associated domain (ZAD)-containing zinc finger proteins based on ZAD protein sequence. Blue labels indicate Drosophila melanogaster proteins, red labels mark Kipferl orthologs in different species. Branches that are supported by an ultrafast bootstrap (UFBoot) value >=95% are indicated by a black dot. Branch lengths represent the inferred number of amino acid substitutions per site, and branch labels are composed of gene name (if available), genus, species, and accession number.

Diagnostic plots for rank 1-5 for the AlphaFold2 Multimer prediction of the Rhino chromodomain with the Kipferl ZnF cluster 1. (A) PAE plot (B) pLDDT plot (C) Superposition on the Rhino chromodomain of the models for rank 1 – 5, as Cα trace.

Individual line graphs depicting SEC-MALS results for the examined Rhino chromodomain constructs with in solution molecular weight measurements depicted in red. (B) Bar graph summarizing circular dichroism spectrum measurements for the tested Rhino chromodomain constructs.