In vitro activity: In cells, A-196 induced a global decrease in H4K20me2 and H4K20me3 and a concomitant increase in H4K20me1. A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a first-in-class chemical probe of SUV4-20 to investigate the role of histone methyltransferases in genomic integrity.

Kinase Assay: A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a first-in-class chemical probe of SUV4-20 to investigate the role of histone methyltransferases in genomic integrity.

Cell Assay: U2OS cells are seeded on 6-well plates with 3 μM A-196 or DMSO as a control, and incubated for 48 h. The cells are washed once in 1 X PBS and then lysis buffer (20 mM Tris-HCl pH 7.5, 0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA, 10 mM MgCl2, PMSF, protease inhibitors, benzonase) is added to half the cells to create whole cell extract (WCE). The remaining cells are subjected to sequential cellular fractionation. First the cell pellet is resuspended in hypotonic buffer A (10 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 0.3 M sucrose, 1 mM DTT and protease inhibitors) and then 0.1% triton X-100 is added. The cells are incubated for 15 min on ice and pelleted by centrifugation at 1,500g. The supernatant is clarified by centrifugation at max speed and saved as the cytoplasmic fraction. The pellet is resuspended in buffer B (3 mM EDTA, 3 mM EGTA, 1 mM DTT and protease inhibitors) and incubated on ice for 40 min and then centrifuged at 1,500g for 5 min. The supernatant is clarified and saved as the nucleoplasmic fraction. The pellet is resuspended in lysis buffer and incubated for 5 min at room temperature before being resuspended in 4× loading dye. The final lysate contains the solubilized chromatin fraction.

Nat Chem Biol. 2017 Mar;13(3):317-324.