Panel A shows a dot plot comparing PML fused with RARA interactome, ATRA signaling, and SUMO pathway proteins. Panel B shows Western blots of HPC7 pull-down and input samples with Retinoic Acid (RA), PML fused with RARA, and controls. Panel C shows a scatter plot comparing PML fused with RARA interactors with arsenic trioxide (ATO)-induced SUMOylome, including a Venn diagram. Panel D shows Western blots of 293T cells analyzing NCoR SUMO2 modification by PML fused with RARA and TAK-981. Panel E shows Western blots comparing SUMO1- and SUMO2-modified NCoR in 293T cells expressing PML fused with RARA.
PML::RARA partners undergo SUMO2 conjugation. (A) Dot plot of the top 30 proteins that showed an increase in interaction with PML::RARA, ranked by fold enrichment over NLS-BioID control. N = 2 for NLS-BioID, N = 3 for PML::RARA-BioID. Proteins annotated in green are classified as ATRA signaling (reported to participate in RA/ATRA signaling based on published literature), and those annotated in pink are “SUMO pathway” proteins (previously implicated in the SUMO pathway or identified as SUMO conjugates). (B) Western blot validation of selected PML::RARA interactors after biotin labeling followed by streptavidin pulldown. Cells were pretreated with or without RA (1 μM) for 2 h, followed by biotin (50 μM) addition, with RA treatment continued for a total of 18 h. N = 3. (C) Top: Venn diagram depicting 21 PML::RARA partner proteins from the HPC7 BioID that were previously found in ATO-responsive SUMOylome from primary mouse APL blasts (Tessier et al., 2022). Bottom: Diagram showing the comparative enrichment of PML::RARA interactants (versus NLS) and ATO-enhanced sumoylation for these 21 proteins. RARA- and PML-binding proteins are highlighted in red and blue, respectively. (D and E) Western blot analysis of lysates and Ni-NTA–purified fractions from 293T cells transfected with the indicated constructs. N = 2. In D, 293 T cells were treated with SUMO inhibitor TAK-981 (100 nM) for 4 h on day 2 after transfection before analysis. NcoR-S: SUMO2-conjugated NCoR. Asterisk denotes remaining unsumoylated NCoR. Source data are available for this figure: SourceData F2.