FIG. 4.

Human TFIIIC has an associated HAT activity. (A) TFIIIC, but not TFIIIB or RNA polymerase III, contains an associated HAT activity. The filter binding HAT assay was performed with 1 μg of core histones, [³H]acetyl-CoA, and 200 ng of highly purified human TFIIIC (lane 1), RNA polymerase III (lane 3), TFIIIB (lane 4), and mock-purified TFIIIC from the M2 agarose affinity column (lane 2). (B and C) TFIIIC acetylates free histones H3 and H4. (B) Core histones were incubated with affinity purified TFIIIC (lane 1), mock immunoprecipitate (lane 2), RNA polymerase III (lane 3), and TFIIIB (lane 4) and then resolved on an SDS–12% polyacrylamide gel that was stained with Coomassie brilliant blue (left) and then analyzed by fluorography (right). (C) Core histones were incubated either with assay buffer (lane 1) or with 50 ng (lane 2), 75 ng (lane 3), or 200 ng (lane 4) of TFIIIC and analyzed as for panel B. (D) TFIIIC acetylates HeLa nucleosomal histones. The HAT assay was performed as described above. Nucleosomes were incubated with 25 ng of recombinant p300 HAT domain (lane 1), mock immunoprecipitate (for TFIIIC) (lane 2), and 0.5 μg of TFIIIC (lane 3). (E) TFIIIC contains three polypeptides with HAT activity. In-gel HAT activity assays were performed following resolution of TFIIIC on SDS–8% polyacrylamide gels containing histones (lane 1) or BSA (lane 2).