Fig. 5: Pit interacts with HP1a via a conserved HP1a interaction motif.

a, Schematic of the proximity labelling experiment (top). In S2R+ cells expressing V5-APEX2–Pit, proteins close to Pit are biotinylated following the addition of biotin-phenol and H₂O₂, and then isolated using streptavidin pull-down. Input lysates and streptavidin eluates from S2R+ cells expressing V5-APEX2–Pit, with and without H₂O₂ treatment, were analysed by western blotting and probed with anti-HP1a showing HP1a as a biotinylated target of V5-APEX2–Pit (bottom). b, Schematic of the co-immunoprecipitation (co-IP) experiment (top). Nuclear lysates from S2R+ cells were incubated with either anti-Pit or control IgG, followed by immunoprecipitation using Protein G magnetic beads. Western blot analysis of input, flow-through and co-IP samples probed with anti-HP1a and anti-Pit (bottom). HP1a (magenta arrow) co-immunoprecipitates with Pit (yellow arrow). c, AlphaFold-Multimer structural prediction of a 25-amino-acid peptide from the C-terminal disordered domain of Pit containing the putative PxVxL HP1a-interacting motif (PVVDLKVGA), with the CSDs of an HP1a dimer (UniProt identifier: P05205). Heatmap indicating the predicted Local Distance Difference Test (pLDDT), a measure of confidence in the predicted structure (top). In the predicted structure, the Pit PxVxL motif binds to the HP1a–CSD dimer cleft, with the central valine residue of PVVDL positioned at zero (bottom). d, Schematic of in vitro binding assay where purified His-tagged Pit^WT or Pit^PxExL was incubated with purified HP1a^WT or the HP1a^I191E dimer mutant and captured using Ni-NTA magnetic beads, and then eluted for analysis (left). Western blots of input and elution fractions in the His-binding assay probed with antibodies to Pit and HP1a (centre). HP1a is enriched in the elution fraction when Pit^WT and HP1a^WT are incubated together (magenta arrow), indicating a direct interaction in vitro. This interaction is lost when Pit^WT and HP1a^I191E or Pit^PxExL and HP1a^WT are incubated together (faded magenta arrow), indicating that Pit binds the HP1a dimer cleft via its PxVxL motif. HP1a band intensities shown as fold enrichment over the background control (beads + HP1a only; right). Background signal was set to 1.0 (dashed line). Bars represent the mean of two independent experiments.

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