MMSET mediates histone H4K20 trimethylation in vivo. (A) MMSET, Suv4-20h2, Suv39H1, and G9a were immunoprecipitated from KMS11 cells and incubated in vitro with histone substrates and ¹⁴C-SAM. Immunoprecipitated β-actin served as a negative control. Specific antibodies to trimethylated histone H4 indicate that both MMSET and Suv4-20h2 trimethylate H4K20. (B) Schematic diagram of the integrated Gal4 reporter gene, arrows show sites corresponding to PCR primers. (C) Luciferase activity from an integrated reporter gene was measured subsequent to transfection with Gal4 DNA binding domain, the C-terminal portion of MMSET (Gal4-MB4-2-II), and known repressors (GAL4-G9a, GAL4-Cbx7). (D) Chromatin configuration of an integrated Gal4 reporter gene in response to Gal4-MMSET. Chromatin from cells transfected with a Gal4-MMSET fusion or the Gal4 DNA binding domain was precipitated with the indicated antibody and a PCR fragment encompassing the Gal4 DNA binding sites and promoter region of the reporter gene was amplified. The fraction of input precipitated by each antibody was normalized to the fraction precipitated by antihistone H3 antibodies. Black bar, transfection with Gal4-DBD; gray bar, transfection with Gal4-MMSET-MB4-II; white bar, transfection with Gal4-MMSET (C terminal domain). The results shown are the average (± SD) of 4 independent experiments. (E) Relative levels of acetylation of H3 and H4 on the Gal4 target gene associated with transfection of Gal4-MMSET as shown by quantitative ChIP. Above the graphs, qualitative ChIP is displayed to show the level of recruitment of wild-type and mutant Gal4-MMSET to the target promoter. Gray bar, transfection with wild-type Gal4-MMSET; black bar, transfection with mutant Gal4-MMSET Y1118A; striped bar, transfection with Gal4. (F) Relative levels of methylated histone lysines on the Gal4 reporter associated with wild-type and mutant Gal4-MMSET as determined by quantitative ChIP was performed for each of the indicated modifications on histones H3 and H4. Promoter occupancy of both wild-type and mutant Gal4-MMSET as tested by immunoprecipitation with antibody to Gal4 is shown. Values represent relative signal versus total histone. Gray bar, transfection with wild-type Gal4-MMSET; black bar, transfection with mutant Gal4-MMSET Y1118A. Error bars represent SD.