Two different Hmr mutations interfere differently with HMR interactome and HMR complex formation (related to Fig 3).

(A) Recombinantly co-expressed HMR^dC and LHR do not interact. Western blot showing anti-HA immunoprecipitation in nuclear extracts from Sf21 insect cells transfected with HA-HMR and His-LHR. IP performed with anti-HA antibody and western blot probed with anti-HMR or anti-LHR antibodies. (B) HMR C-terminus is required for HMR interaction with LHR and HP1a in D. mel SL2 cells. Western blot showing HMR immunoprecipitation in SL2 cells stably transfected with either full length HMR or a C-terminally truncated HMR^dC along with Myc-LHR. IP performed with anti-FLAG antibody targeting FLAG-HMR and western blot probed with anti-HA (HMR), anti-Myc (LHR) and anti-HP1a. (C) Western blot showing LHR immunoprecipitation in SL2 cells stably transfected with either full length HMR or a C-terminally truncated HMR^dC along with Myc-LHR. IP performed with anti-Myc antibody targeting Myc-LHR and western blot probed with anti-FLAG (HMR) and anti-LHR. (D) Volcano plot highlighting interactions depleted in HMR^dC. X-axis: log₂ fold-change of FLAG-HMR^dC IPs (right side of the plot) vs FLAG-HMR⁺ IPs (left side of the plot). Y-axis: significance of enrichment given as–log₁₀ p-value calculated with a linear model. HMR complex subunits are labelled in red. In blue are factors depleted upon HMR^dC mutation (among the endogenous or overexpression-induced interactions of HMR). Unlabeled additional bait-specific interactors are listed in S3 Table. (E) GO terms depleted upon HMR^dC mutation. (F) Volcano plot highlighting interactions depleted in HMR². X-axis: log₂ fold-change of FLAG- HMR² IPs (right side of the plot) vs FLAG- HMR⁺ IPs (left side of the plot). Y-axis: significance of enrichment given as–log₁₀ p-value calculated with a linear model. HMR complex subunits are labelled in red. In blue are factors depleted upon HMR² mutation (among the endogenous or overexpression-induced interactions of HMR). Unlabeled additional bait-specific interactors are listed in S3 Table. (G) GO terms depleted upon HMR² mutation. In (D) and (G) proteins labelled or used for GO search include endogenous or overexpression-induced interactions of HMR (i.e. enriched in HMR⁺ or HMR vs CTRL with p < 0.05) and differentially enriched between HMR⁺ and the HMR mutant analyzed (log₂ fold-change (HMR*/HMR+) < 1.5).

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