Yeast three-hybrid screen. (a) Illustration of two-component bait plasmid LexA-CREB-YeACBP. LexA-CREB (containing the activation domain of CREB, aa 1 to 283, fused to the LexA DNA binding domain) and a nucleus-localized fragment of CBP (representing the CREB binding domain, aa 461 to 682) were cloned into plasmid pBTMYeA, a derivative of pYeA. The bait plasmid is ampicillin resistant and enables Trp production. P, promoter; T, terminator; NLS, nuclear localization signal. (b) Interaction of VP16-RbAp48 with the CREB-CBP complex and CBP alone but not with CREB. LexA-CBP, LexA-CREB, and LexA-CREBM1 are LexA fusion constructs encoding aa 461 to 682 of CBP, aa 1 to 283 of CREB, or aa 1 to 283 of CREB with a Ser₁₃₃Ala mutation, respectively. LexA-CREBM1-YeACBP is a two-component bait plasmid identical to LexA-CREB-YeACBP with the exception of the described Ser₁₃₃Ala mutation. The levels of interaction with VP16-RbAp48, indicated by the plus and minus signs, were determined by assaying the growth of the transformants on a His-negative background.

Augmentation of RbAp48-CBP interaction by phosphorylated CREB. (a) Direct binding of RbAp48 and RbAp46 to CBP. GST pull-down assays were performed with GST or GST-CBP_551–682 and recombinant His-tagged RbAp48 or -46. RbAp48 or -46 bound to GST-CBP_551–682 was visualized by Western blotting. (b) Binding of RbAp48 to the phosphorylated CREB-CBP complex. GST- or GST-CBP_551–682-coupled beads were incubated with His-tagged RbAp48 in the presence of nonphosphorylated (+CREB) or phosphorylated (+pCREB) CREB. The binding of RbAp48 and CREB to GST-CBP_551–682 was visualized by Western blotting.

Blockage of histone binding to the CBP-RbAp48 complex by acetylation. Core histone octamers (a) and mononucleosomes (b) were acetylated by CBP. GST pull-down assays were performed using GST or GST-CBP_551–682 in the presence or absence of RbAp48 with acetylated versus underacetylated histone particles. (c) Both hypoacetylated (underacetylated) and CBP-acetylated (acetylated) core histones or mononucleosomes were incubated with GST- or GST-RbAp48-coupled beads directly. Bound core histone octamers were assayed by Western blotting using an antibody specific for histone H4 (histones). Bound mononucleosomes were detected by two antibodies against either H3 or H4 (mononucleosomes).

Interaction between RbAp48 and CBP in HeLa cells. (a) GST pull-down assay. A GST fusion protein encoding RbAp48_273–425, which contains carboxyl-terminal WD repeats 4 through 7 (GST-RbAp48_273–425), and a GST fusion protein containing full-length RbAp48 (GST-RbAp48) were coupled to glutathione-agarose beads and incubated with HeLa cell nuclear extracts. CBP bound to the GST-RbAp48 fusion proteins was visualized by Western blotting. (b) Association of CBP and RbAp48 in vivo. Endogenous RbAp48 was immunoprecipitated from HeLa cell nuclear extracts using a monoclonal antibody against RbAp48. CBP coimmunoprecipitated with RbAp48 and was visualized by Western blotting. Normal mouse immunoglobulin G (IgG) was used as a negative control.

Bridging of CBP to histone proteins by RbAp48. GST- or GST-CBP_551–682-coupled beads were incubated with chicken core histones (a) or mononucleosomes (b) in the presence or absence of full-length RbAp48. Bound core histone octamers were assayed by Western blotting using antibody specific for histone H4 (core histones). Bound mononucleosomes were detected by two antibodies against either H3 or H4 (mononucleosomes).

Facilitation of p300-mediated transcription of a chromatinized template by RbAp46. (a) In vitro transcription assays performed using Gal-VP16-remodeled chromatin templates. The DNA template contains five Gal4 DNA binding sites upstream from the adenovirus MLP and a 390-nt G-free transcription cassette. Addition of AcCoA alone or p300 and AcCoA did not significantly enhance transcription. Addition of p300 and RbAp46 modestly activated transcription, while addition of p300, RbAp46, and AcCoA significantly increased the transcription levels of the 390-nt transcript. Similar results were obtained in four independent assays. (b) In vitro transcription assays using a naked DNA template. The DNA template contains five Gal4 DNA binding sites upstream from the adenovirus MLP and a 390-nt G-free transcription cassette. As an RNA recovery control, a DNA template encoding a 170-nt G-free transcription cassette placed downstream from the adenovirus MLP was used in the productive-transcription assays. When results were normalized with the MLP control, no significant increase in transcription was detected among the different manipulations. These results were obtained in three independent assays.