Figure 3.

Chromatin association is required for TRIM24 SUMOylation. A, HDAC inhibitor treatment promotes TRIM24 SUMOylation. HEK293T cells were treated with TSA (2 μm) for 60 min, followed by histone extraction (right) and WCL (left) preparation and subjected to Western blot analysis using the indicated antibodies. B, HEK293T cells were transfected with the indicated FLAG-TRIM24 WT and mutant protein-expressing plasmids. 24 h post-transfection, cells were treated with TSA for 60 min, and WCL were subjected to Western blot analysis with FLAG and actin antibodies. C, HDAC inhibition promotes chromatin association of TRIM24. DMSO- and TSA-treated MCF7 cells were fractionated to prepare cytosol (Cy), nuclear (Nu), and chromatin (Ch) fractions. Western blot analysis was performed using the indicated antibodies. Lamin B and GAPDH serve as positive controls for chromatin and cytosol fractions, respectively. TRIM28 serves as a loading control for comparison of individual fractions between DMSO and TSA treatment. D, HEK293T cells treated with TSA and WCL were subjected to Western blot analysis with the indicated antibodies. E, IACS-9571 dissociates TRIM24 from chromatin and down-regulates its SUMOylation. HEK293T cells treated with DMSO, TSA, or 9571 for 60 min were used to generate WCL, soluble (cytosol and free nuclear), and chromatin-bound fractions. Western blot analysis was performed using the indicated antibodies. F, MCF7 cells were treated with the indicated concentrations of BRPF1 inhibitor, GSK 5959, or IACS-9571 for 24 h. WCL were subjected to Western blot analysis with the indicated antibodies.