(A) Predicted amino acid sequence of hTFIIIC90 and sequence alignments with a hypothetical yeast protein. The peptide sequences obtained by microsequence analyses are underlined. Two potential zinc finger motifs are doubly underlined. (B) Identification of the cDNA-encoded protein as the 90-kDa hTFIIIC2 subunits. The polypeptide components of a highly purified TFIIIC2 preparation were analyzed in a silver-stained gel (lane 1). Anti-hTFIIIC90 antibodies were used to detect hTFIIIC90 in a highly purified TFIIIC2 preparation (lane 2) and in a purified baculovirus-expressed FLAG-hTFIIIC90 (lane 3).

Immunoprecipitation of the TFIIIC2 complex and immunodepletion of TFIIIC transcription activity by anti-hTFIIIC90 antibodies. (A) Immunoprecipitates from HeLa nuclear extracts treated with preimmune (P) and immune (I) anti-hTFIIIC90 antibodies in BC500–0.1% NP-40 were subjected to Western blot analysis. The polypeptides were detected by a mixture of antibodies against hTFIIIC220, -110, -102, -90, and -63. (B) Nuclear extracts treated with preimmune (P) and immune (I) anti-hTFIIIC90 antibodies were used for in vitro transcription assays with the 5S, VAI, and tRNA templates (lanes 1 to 6) and with the pG₅HMC₂AT and pMLΔ53 templates (lanes 11 to 14). The immunodepleted extract employed for lanes 1 to 6 retained a small amount (<10%) of hTFIIIC90 (data not shown). The immunodepleted extract employed for lanes 7 to 10 had no detectable hTFIIIC90 (data not shown), and immunopurified TFIIIC (the same preparation analyzed for Fig. 1B) was added in lanes 9 (1 μl) and 10 (2 μl).

Interactions of hTFIIIC90 with subunits of TFIIIC2 and TFIIIB and an initiation-specific RNA polymerase III subcomplex. Input samples contained 10% of the amounts used for the interactions. (A) Reticulocyte lysates containing expressed ³⁵S-hTFIIIC220, -hTFIIIC110, -hTFIIIC102, -hTFIIIC63, or -hTAF_II100 were incubated with M2 agarose-immobilized FLAG-hTAF_II80 or M2 agarose-immobilized FLAG-hTFIIIC90, and samples were washed, eluted, and analyzed as described in Materials and Methods. (B) Reticulocyte lysates containing expressed ³⁵S-hTFIIIC90 were incubated with M2 agarose-immobilized hTAF_II80 or M2 agarose-immobilized FLAG-hTFIIIB90 and with glutathione Sepharose-immobilized GST, GST-TBP, GST-RPC62, GST-RPC53, GST-RPC39, GST-RPB6, or GST-RPB5, and samples were washed, eluted, and analyzed as described in Materials and Methods. The amounts of the immobilized tagged recombinant proteins were normalized by SDS-PAGE with Coomassie blue staining. Equivalent amounts of the various FLAG-tagged proteins or the GST fusion proteins were bound to M2 agarose or glutathione Sepharose, respectively.

Recombinant hTFIIIC90 possess HAT activity. (A) FLAG epitope-tagged hTFIIIC90 was expressed in Sf21 cells and purified by M2 agarose affinity chromatography. The protein was resolved by SDS–8% PAGE and either subjected to staining (lane 1) or Western blotting (lane 2). (B to D) Baculovirus-expressed hTFIIIC90 and hTAF_II80 samples were electrophoresed in an SDS–8% polyacrylamide gel containing either no protein (B), histones (C), or bovine serum albumin (D) and subjected either to silver staining (B) or to an in-gel HAT activity assay (C and D).

Substrate specificity of hTFIIIC90 HAT activity. (A and B) Baculovirus-expressed hTFIIIC90 shows a strong HAT activity with highly purified HeLa core histones. The HAT activity assay was performed with 1.5 μg of HeLa core histones, [³H]acetyl-CoA, and 25 ng of the p300 HAT domain (lane 1), 200 ng of TFIIIC (lane 2), 200 ng of hTFIIIC90 (lane 3), 200 ng of hTAF_II80 (lane 4), or mock immunoprecipitate (lane 5) and subjected to filter binding assay (A) or SDS-PAGE to assess the substrate specificity. The gels were stained with Coomassie brilliant blue (top) and then analyzed by fluorography (bottom) (B and C). (C) hTFIIIC90 acetylates HeLa nucleosomal histones. The HAT assay was performed as described above. Native HeLa nucleosomes were incubated with 25 ng of recombinant p300 HAT domain (lane 1), mock immunoprecipitate (for hTFIIIC90) (lane 2), 0.5 μg of TFIIIC (lane 3), 200 ng of purified recombinant hTFIIIC90 (lane 4), and 200 ng of purified TAF_II80 (lane 5).

Site specificity of hTFIIIC90 histone H3 acetylation. (A) The sequences of the peptides used for panel B are shown. Sites where ɛ-N acetyllysine was incorporated during peptide synthesis in order to mimic sites that are acetylated in vivo are indicated by Ac. Peptides H3 and the diacetylated H3(9/14) are straight chain, and H3* and H3*(9/18) were synthesized by the multiple antigen peptide method. (B) Histone H3 amino-terminal peptides were incubated either with only FLAG-elution buffer (lanes 1, 3, 5, and 7) or with highly purified hTFIIIC90 (lanes 2, 4, 6, and 8). Acetylation of the peptides was assessed by measuring [³H]acetate incorporation by using the filter binding assay.