Interaction of CBP and PCAF with CDP/cut. (A) Immunoprecipitation/Western blot analysis of transfected HA-tagged CBP and CDP/cut. HeLa cells were cotransfected with expression vectors for CBP and CDP/cut. Immunoprecipitations with antibody or antiserum directed to either the HA-tag (αHA) or CDP/cut, respectively, from the lysates of transfected cells were recovered and subjected to immunoblotting by antisera indicated under the blot. Arrows indicate the predicted size of the detectable immunoblotted protein. (B) [³⁵S]-labeled CDP/cut was synthesized in vitro and mixed with purified FLAG-tagged CBP, corresponding to the residues of CBP as indicated, immunoprecipitated with anti-FLAG M2 antibody (Sigma), electrophoresed, and detected by autoradiography. (Lower) [³⁵S]-labeled CDP/cut was synthesized in vitro and mixed with purified FLAG-tagged PCAF (wild type) or PCAF (Δ579–608) and visualized as in Upper. (C) Acetylation of CDP/cut in vivo. Lysates from HeLa cells, transfected with pMT₂-CDP, were immunoprecipitated with anti-CDP/cut antiserum or preimmune serum as a control. Immunoprecipitates were analyzed by immunoblot analysis by using a polyclonal antiacetylated lysine antibody.

CDP/cut is regulated by PCAF HAT activity through sequences between the C3 and HD and corresponds to the acetylation of CDP/cut in vivo. (A) Schematic diagram depicts various deletion mutants of the CDP/cut cDNA fused in frame to the DBD of GAL4. The deletion mutants of CDP/cut cDNA were cloned into parental plasmid pM (CLONTECH) containing the DBD of GAL4. The human CDP/cut cDNA fragments, fused to GAL4, were tested in a transactivation assay and monitored for the repression of the cotransfected target CAT reporter gene, GAL4InrCAT, as shown in the photo (Right). The NULL (A Upper) sample corresponds to the background expression of the reporter gene construct GAL4InrCAT. (B) Comparison of the relative activities of the individual CDP/cut cDNAs shown (A) fused to the GAL4 DBD is shown in the presence (or absence) of the cotransfected PCAF HAT activity. Results compare the ability of specific CDP/cut deletions to repress activity of the GAL4InrCAT target. Full-length wild-type CDP/cut [pG4-CDP (wild type)] and deletion mutant of CDP/cut (pG4-CDPΔ1186–1256) are compared with the background level of CAT activity rendered in the presence of the parental GAL4 expression vector shown as pGAL4 (n = 10). (C) To analyze the in vivo acetylation of GAL4 derived fusion proteins of CDP/cut, G4-CDP (wild type) and G4/CDPΔ1186–1256, 8 μg of PCAF (wild type) plasmid was cotransfected with 5 μg of either pG4-CDP (wild type) or pG4/CDPΔ1186–1256 into 293 cells. Acetylation of the CDP/cut fusions with the DBD of the GAL4 protein was determined by immunoprecipitation of equivalent amounts of G4/CDP (wild type) and G4/CDPΔ1186–1256 protein with a mouse monoclonal anti-GAL4 antibody and precipitated with goat anti-mouse IgG agarose. The immunoprecipitates, separated by SDS/PAGE, were immunoblotted with antiacetylated lysine antibodies (Upper). A corresponding immunoblot (Lower) by using an anti-GAL4 mAb was performed with the identical cell lysates [as shown (Upper)] from 293 cells, cotransfected with PCAF and either the parental GAL4 vector pM, pG4-CDPΔ1186–1256, or pG4-CDP (wild type).

CDP/cut is a target of acetylation by the HAT PCAF. (A) In vitro acetyltransferase reactions were used on in vitro-synthesized and unlabeled CDP/cut. Equivalent molar amounts of CBP (wild type), PCAF (wild type), and PCAF (Δ579–608) were immunoprecipitated from cells expressing HA-tagged CBP (wild type), FLAG-tagged PCAF (wild type), and FLAG-tagged PCAF (Δ579–608) and used in an in vitro reaction along with [¹⁴C]acetyl-CoA where indicated. CDP/cut was synthesized with [¹⁴C]leucine in vitro and immunoprecipitated as a control. Immunoprecipitated products were resolved on a SDS/PAGE gel and autoradiographed. Lower corresponds to an immunoblot of the in vitro-translated product of CDP/cut as the input amount for protein acetyltransferase reactions in vitro. (B) Acetylation of C3 and HD of CDP/cut. GST fusion protein of CDP/cut (C3-HD) was affinity purified and subjected to acetylation reactions with the same FLAG-tagged reagents described above. Acetylated product of the GST fusion with CDP/cut was separated by SDS/PAGE and autoradiographed. Free histone H3 served as a positive control for the acetyltransferase activity of PCAF. Gels were stained with Coomassie stain for estimation of protein levels before autoradiography (not shown). (C) Acetylation of CDP/cut disrupts DNA binding to the TK promoter. EMSA of the acetylated product of GST-CDP/cut (C3-HD) after in vitro acetyltransferase reactions as described above with the exception that reactions were performed with unlabeled acetyl-CoA.

Localization of PCAF acetyltransferase activity on CDP/cut. (A) A schematic diagram depicts the location of two synthetic peptides used in the competition assay relative to the structure of CDP/cut, between C3 and HD in a GST fusion protein. The primary sequence of residues for the competing peptides is shown (Lower). (B) Consensus site of residues within the sequence derived from peptide 1. Sequence comparison of residues located within the histone-like consensus of CDP/cut and sequences of the histone H4 from man and mouse. (C) Peptide competition for acetyltransferase activity was performed with CDP/cut and PCAF. Synthetic peptides shown in A were used in an in vitro protein acetyltransferase assay as competing substrates with immunoaffinity-purified FLAG-tagged PCAF corresponding to the wild-type PCAF in the presence of [³H]acetyl-CoA. Synthetic nonacetylated peptides 1 and 2, preacetylated peptide 1 [100 mol/mol of GST-CDP (C3-HD)] or a synthetic peptide corresponding to the 24 amino terminal of mouse histone H4 were introduced into acetyltransferase reaction as substrates before the addition of GST-CDP (C3-HD). The GST-CDP (C3-HD) fusion protein was introduced into the acetyltransferase reactions and quenched with unlabeled acetyl-CoA after 10 min. Incorporation of [³H]acetyl-CoA into GST-CDP (C3-HD) was determined by filter-binding assays. (D) EMSA of acetylated GST-CDP (C3-HD). Acetyltransferase assay was performed as described in C with the exception that the acetyltransferase reactions with GST-CDP (C3-HD) were conducted with unlabeled acetyl-CoA. After the termination of the acetyltransferase reactions conducted in vitro, GST-CDP (C3-HD) was affinity purified with glutathione–agarose and eluted with 10 mM glutathione. Affinity-purified GST-CDP (C3-HD) products of the acetyltransferase reactions were then added to DNA-binding reactions by using the [³²P]-labeled TK2C oligonucleotide, and reactions were loaded onto 4% polyacrylamide gels for EMSA.