Extended Data Fig. 6: Pit interacts with HP1a via a conserved PxVxL HP1a-interacting motif.

a, Top: Schematic of the V5-APEX2–Pit construct used to generate a stable S2R+ cell line for biotin-based proximity ligation. Bottom left: Validation of APEX2-tagged Pit expression using anti-V5 immunostaining. After treating cells with H₂O₂, biotinylation was confirmed using fluorescent streptavidin staining. Bottom right: Western Blots of inputs and streptavidin-captured (biotinylated) proteins following APEX2–Pit proximity labelling, probed with anti-Streptavidin antibody. b, Sequence alignment showing conservation of a PxVxL motif in Pit within Drosophila species (top) and across non-Drosophila eukaryotic model organisms (bottom). c, Left: Western Blot probed with anti-Pit to confirm expression of His-tagged recombinant Pit in E. coli after IPTG induction. Right: SDS–PAGE analysis of proteins eluted from Ni-NTA resin after purification of recombinant Pit in Pit^WT and Pit^PxExL forms. Eluted proteins (left lane) were further purified using size-exclusion chromatography (right lane). d, His-tagged binding assay was performed using 5 µM His-tagged Pit protein (WT or PxExL mutant form) incubated with decreasing concentrations of HP1a (WT or I191E dimer-mutant form) at 50, 25, 10, and 5 µM. As a negative control, HP1a alone (no Pit protein) was incubated with beads, revealing non-specific binding of HP1a to the Ni-NTA magnetic beads (brown arrows). Thus, a dilution series of Beads + HP1a alone was included to assess background signal. Dilution series reveals an enrichment for HP1a signal (solid magenta arrow) over background (brown arrow) when Pit^WT and HP1a^WT are incubated at equimolar concentrations (5 µM). This enrichment is reduced when Pit^WT and HP1a^I191E or Pit^PxExL and HP1a^WT were incubated (faded magenta arrow).

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