FIG. 8.
Swapping critical Bcl-6 charged pocket residues alters transcriptional repression and corepressor binding properties. Mutant Bcl-6 BTB domains were generated by swapping the K47 residue for arginine (as in PLZF) or serine (as in FAZF). (A) Reporter assays in 293T cells transfected with 25 ng of GAL1-147 or GAL-Bcl-6BTB mutants as indicated along with 100 ng of reporter and 5 ng of the internal control. Results are tabulated as fold repression compared to that of GAL1-147 normalized to the internal control. (B) Mammalian two-hybrid assays in 293T cells transfected with 25 ng of GAL-Bcl-6 bait and 150 ng of VP-16-N-CoR or -SMRT prey or 5 ng of GAL-Bcl-6 bait with 25 ng of VP16-B-CoR prey in HeLa cells. Results are reported as fold activation of the (GAL)5-tk-Luc reporter construct. B-CoR interaction was also tested in 293 T cells with similar results (not shown). (C) Coimmunoprecipitations (IP) were performed with in vitro-translated BTB domains and N-CoR as described previously. The top section shows fluorography of BTB domains precipitated with N-CoR antibodies, and the bottom section shows the appropriate lysates prior to immunoprecipitation.