Supplementary Figure Legends
Figure S1. H3 ChIP-qPCR over T1 and BX2 transgenes.
H3 signal on T1 and BX2 as measured by ChIP-qPCR on three different regions of the
lacZ sequence shown on Fig. 1A. Signal was normalized to input and compared to
signal at the same control genomic region (chr2R: 2336913-2337023) used in Fig. 1BD. Error bars represent the standard error of the mean between two technical replicates.
Figure S2. Analysis of the ping-pong processing.
An equal number of piRNA reads mapping to the P{lArB} transgene in each library was
sampled 1000-times. Shown are the distributions of the fractions of reads participating
in ping-pong piRNA pairs.
Figure S3. piRNA sequences that mapped to BC69 target locus.
The junction between hsp70 and rosy segments is present in BC69, but not in P1152.
Figure S4. Sequence alignment of the Rhino chromodomain.
(A) Structure based sequence alignment of HP1 chromodomain from D. melanogaster.
The secondary structure of Rhino is shown on the top of alignment. The three
conserved aromatic residues involved in methyl-lysine recognition are highlighted with
asterisk at the bottom of the alignment.
(B) Alignment of the Rhino chromodomain with HP1 family chromodomains from
human, mouse, Drosophila and S. pombe. The three conserved aromatic residues
involved in methyl-lysine recognition are marked with arrows.
Figure S5. Genome-wide analysis of the correlation between Rhino and H3K9me3
occupancy. Shown are Rhino ChIP-Seq enrichments (RPMs, ChIP/Input) for genomic
regions that have different level of H3K9me3 mark. The analysis was performed on 50
kB genomic windows that were binned into four groups according to the level of
H3K9me3 ChIP-Seq enrichment.
Figure S6. Recognition of the H3K9me3 peptide by the Rhino chromodomain. (A)
Superposition of the H3K9me3 peptide-bound Rhino chromodomain (in color) with the
free form chromodomain (in silver) reveals no conformational change. (B) The K9me3
group of the H3 peptide is recognized by three conserved aromatic residues.
Figure S7. Intermolecular recognition contacts at the dimer interface of the RhinoH3(1-15)K9me3 complex. (A) Hydrophobic interactions at the dimer interface. (B)
Hydrogen bonding interactions at the dimer interface.
Figure S8. Rhino and Cutoff form a complex in vivo.
Co-immunoprecipitation from ovary extract shows specific interaction between tagged
Rhino-BioTap and Cutoff-eGFP proteins.
Figure S9. Association of Cutoff with piRNA cluster chromatin depends on RNA.
Cutoff signal as measured by ChIP-qPCR on several regions of cluster 42AB and
control region cluster 20A (n=2). Signal was normalized to input and compared to signal
at a control genomic region (rp49 gene). Where indicated, RNase treatment was
performed on the lysate prior to immunoprecipitation.
Figure S10. Size profile of small RNAs generated from the 42AB piRNA cluster.
Total small RNAs of the size range between 18 and 29 nt were cloned from Drosophila
ovaries. The peak at 21 nt corresponds to siRNAs. The broader peak at 23-28nt
corresponds to piRNAs.
Figure S11. GRO-Seq analysis of transcriptional output from double-stranded
piRNA clusters upon Rhino depletion.
Shown are RPM (reads mapping to the piRNA cluster normalized to total number of
mapped reads in the respective library) of GRO-Seq on ovaries from wildtype and Rhino
knockdown flies.
Figure S12. Specification of piRNA clusters by trans-generationally inherited
piRNAs in interspecies hybrids between D. melanogaster and D. simulans. Shown
are piRNAs (RPMs) expressed in ovaries of D. melanogaster, D. simulans, and F1
hybrids between D. melanogaster females and D. simulans males that inherited the
piRNAs from their mothers. The left panel shows piRNAs mapping to a D. simulans
piRNA cluster (chr2L:15156-36342), and the right panel shows mapping of piRNAs to a
D. melanogaster piRNA cluster (chr3L:23257666-23337605). The D. melanogaster
piRNA cluster is expressed in the F1 progeny (F1), but the D. simulans piRNA cluster is
not.