Panel A shows a line graph depicting colony counts from serial replating assays of primary mouse Lin- hematopoietic progenitors transformed with UBC9/RARA, RARA fusions, or their I396E mutants defective for NCoR binding. The x-axis represents the replating number (MC 2, 4, 5, and 6), and the y-axis represents the colony number. Different lines represent different transformations, with UBC9/RARA showing a significant decrease in colony number over time. Panel B shows a line graph of FACS analysis of differentiation marker Gr-1 expression in Lin- progenitors transformed with indicated fusions at the specified replating. The x-axis represents the replating number (MC 2, 4, and 6), and the y-axis represents the percentage of Gr-1 positive cells. Panel C shows a line graph of FACS analysis of stemness marker c-Kit expression in Lin- progenitors transformed with indicated fusions at the specified replating. The x-axis represents the replating number (MC 2, 4, and 6), and the y-axis represents the percentage of c-Kit positive cells. Panel D shows Western blot analysis of lysates and Ni-NTA-purified His-tagged SUMO conjugates from Lin- progenitors. The blots display various proteins, including NCoR, IRF2BP2, GPS2, GATA2, KAP1, and RARA, with different lanes representing different transformations. Panel E shows Western blot analysis of lysates and Ni-NTA-purified His-tagged SUMO conjugates from primary MLL-ENL blasts transduced with RARA fusions. The blots display various proteins, including NCoR, IRF2BP2, GPS2, KAP1, and RARA, with different lanes representing different transformations. Panel F shows bar graphs of RARE/DR5 luciferase reporter assays showing transcriptional activity of RARA, PML fused to RARA, and their mutants after 12 hours of ATRA treatment or vehicle treatment. The x-axis represents different transformations and treatments, and the y-axis represents relative luciferase activity. The graphs indicate significant differences in transcriptional activity between different transformations and treatments.
Fusion of UBC9 to RARA enforces sumoylation of RARA-bound partners and allows long-term progenitor immortalization. (A) Colony counts from serial replating assays of primary mouse Lin− hematopoietic progenitors transformed with UBC9/RARA, RARA fusions, or their I396E mutants defective for NCoR binding. N = 3. (B and C) FACS analysis of differentiation marker Gr-1 (B) or stemness marker c-Kit (C) expression in Lin− progenitors transformed with indicated fusions at the specified replating. N = 2. (D and E) Western blot analysis of lysates and Ni-NTA–purified His-tagged SUMO conjugates from Lin− progenitors (D, N = 3) or primary MLL-ENL blasts transduced with RARA fusions (E, N = 2). PML::RARA-S or UBC9-RARA-S denotes SUMO-conjugated PML::RARA or UBC9/RARA. The asterisk indicates an unidentified posttranslational modification of RARA. (F) RARE/DR5 luciferase reporter assay showing transcriptional activity of RARA, PML::RARA, and their mutants after 12-h ATRA (1 μM) or vehicle (untreated, “Unt”) treatment (N = 2). (A–C) Cells were replated every 5–7 days; MC II, IV, V, and VI denote the second, fourth, fifth, and sixth methylcellulose replating. (A–C and F) Data are presented as the mean ± SD of three technical triplicates from one representative experiment out of at least two biologically independent experiments. Statistical significance was assessed by two-way ANOVA (A–C) or two-tailed unpaired t test (F), ***P < 0.001. Source data are available for this figure: SourceData F4.