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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2014 Mar 10;111(12):E1072–E1081. doi: 10.1073/pnas.1319122111

Binding of the histone chaperone ASF1 to the CBP bromodomain promotes histone acetylation

Chandrima Das a,1, Siddhartha Roy a,b,2, Sarita Namjoshi a, Christopher S Malarkey b, David N M Jones b, Tatiana G Kutateladze b, Mair E A Churchill b, Jessica K Tyler a,3
PMCID: PMC3970516  PMID: 24616510

Significance

The Creb-binding protein (CBP) transcriptional coactivator contains a histone acetyl transferase domain and a bromodomain. Bromodomains bind to acetylated lysines, and their function as previously understood was limited to mediating recruitment to chromatin via binding to acetylated proteins. Here we show that the acetyl lysine-binding activity of the CBP bromodomain has unexpected roles in CBP-mediated acetylation of nonchromatin bound histones, and we show that the interaction between a bromodomain and acetyl lysine is stimulated by autoacetylation. Furthermore, we find that the histone chaperone anti-silencing function 1 binds to the bromodomain of CBP to present free histones correctly for efficient acetylation. Through a combination of structural, biochemical, and cell-based analyses, these studies enhance our understanding of bromodomain function and regulation.

Abstract

The multifunctional Creb-binding protein (CBP) protein plays a pivotal role in many critical cellular processes. Here we demonstrate that the bromodomain of CBP binds to histone H3 acetylated on lysine 56 (K56Ac) with higher affinity than to its other monoacetylated binding partners. We show that autoacetylation of CBP is critical for the bromodomain–H3 K56Ac interaction, and we propose that this interaction occurs via autoacetylation-induced conformation changes in CBP. Unexpectedly, the bromodomain promotes acetylation of H3 K56 on free histones. The CBP bromodomain also interacts with the histone chaperone anti-silencing function 1 (ASF1) via a nearby but distinct interface. This interaction is necessary for ASF1 to promote acetylation of H3 K56 by CBP, indicating that the ASF1–bromodomain interaction physically delivers the histones to the histone acetyl transferase domain of CBP. A CBP bromodomain mutation manifested in Rubinstein–Taybi syndrome has compromised binding to both H3 K56Ac and ASF1, suggesting that these interactions are important for the normal function of CBP.

Chromatin is the physiological template for all genomic processes. The histone proteins that package the DNA into chromatin are subject to posttranslational modifications, including acetylation, methylation, phosphorylation, ubiquitination, and sumoylation, that serve to regulate DNA-templated phenomena such as transcription, replication, repair, and recombination (1). Many histone posttranslational modifications mediate their function by interacting specifically with and recruiting “reader” modules of multifunctional proteins, which often themselves have activities that subsequently further modify the chromatin structure to make the DNA either more or less accessible. For example, the bromodomain is the specific reader module for acetylated lysines on histones and nonhistone proteins (reviewed in ref. 2), where acetylation is one of the most abundant posttranslational modifications in human cells.

The bromodomain is found in many transcriptional coregulators and histone-modifying complexes, including histone acetyl transferases (HATs), enzymes that themselves mediate acetylation. Structural studies have revealed that bromodomains have a conserved structural fold that consists of a left-handed four-helix bundle and two interspersed ZA and BC loops which constitute the active acetyl lysine-binding pocket (3). Despite this conserved overall structure, different bromodomains recognize distinct acetylated lysines in different proteins because the specific amino acid residues within the loops of each bromodomain are critical for determining the acetyl lysine-binding specificity (4, 5).

The general theme for bromodomain function is that they serve to anchor the bromodomain-containing protein to acetylated chromatin templates or to acetylated transcriptional activators. For example, the bromodomains of the yeast ATP-dependent nucleosome remodeler Swi2 and the HAT GCN5 are required for anchoring these chromatin-modifying complexes to acetylated chromatin templates in vitro (6). In other cases, the interaction of bromodomains with non-histone acetylated proteins is important. For example, BRD4, a member of the bromo and extra terminal (BET) subfamily of bromodomain-containing proteins, interacts with the acetylated RelA subunit of NF-κB to promote transcriptional activation of inflammatory genes (7). BRD3 has been shown to interact with the acetylated transcription factor GATA1, recruiting this transcription factor to both active and repressed target genes in a histone acetylation-independent fashion (8). Accordingly, there much interest in generating inhibitors to block the interaction between bromodomains and their acetylated binding partners and thus to reverse changes in gene expression in human disease states.

The Creb-binding protein (CBP) is an important bromodomain containing a transcriptional coactivator that functions as a HAT (9). The transactivation function of CBP is mediated by Zinc-finger domains CH1 and CH3, whereas the KIX and p160 domains serve as platforms to interact with several transcriptional activators (10). The general model for the transcriptional coactivator function of CBP is one of physical recruitment to promoters and enhancers via interactions with transcriptional activators. At the DNA, CBP can acetylate the chromatin-bound histones in the immediate vicinity, causing the chromatin to adopt a more open and accessible state that facilitates transcription. A PHD finger is an integral part of the HAT domain of CBP and also is important for the acetylation function of CBP (11). Autoacetylation of the closely related protein p300 is crucial for its HAT function (12), but the importance of autoacetylation has not yet been shown for CBP. Adjacent to the HAT domain, CBP also possesses a bromodomain that interacts with acetylated histones and nonhistone proteins with varying binding preferences. There is strong evidence for an important functional role for the interaction of the CBP bromodomain with acetylated transcriptional activators [e.g., acetylated p53 (13)]. CBP acetylates p53 on lysine 382, which subsequently is bound by the CBP bromodomain, leading to CBP recruitment (via the bromodomain) to promote p53-mediated gene activation that ultimately determines the cellular responses to stress in the forms of senescence, cell-growth arrest, or apoptosis (13–16). Although the isolated bromodomain of CBP can bind to acetylated histones, including H3 K36 and H4 K20 (5), in vitro, the physiological relevance of the CBP bromodomain binding to acetylated histones is not clear. The closest hint at a function for the interaction between acetylated histones and the CBP bromodomain was provided by the finding that the CBP bromodomain can increase the ability of CBP to acetylate nucleosomal histones in vitro; however, this interpretation is not straightforward, because this function of the CBP bromodomain also was dependent on the interaction between CBP and the Epstein–Barr virus-encoded transcriptional transactivator Zta (17).

In addition to its role in acetylating chromatin-bound histones, we previously had shown that CBP also acetylates non–chromatin-bound histones, specifically on lysine 56 of histone H3 in Drosophila melanogaster and human cells (18). At least in yeast, the histone modification H3 K56Ac (histone H3 acetylated on lysine 56) plays an important role in the delivery of free histones to the replication-dependent histone chaperones so as to drive chromatin assembly following DNA synthesis (19, 20). Before being delivered to the replication-dependent histone chaperones, the free histones are all sequestered by the ubiquitous histone chaperone anti-silencing function 1 (ASF1). We had shown previously that human ASF1 is essential for the CBP-mediated acetylation of free histones on H3 K56 in cells, leading us to propose that ASF1 may physically present the free histones to CBP for acetylation (18). However, the mechanism whereby ASF1 presents free histones to CBP for acetylation was unknown. Once incorporated into the chromatin, the nucleosomes carrying K56Ac have a looser intrinsic structure (21, 22), promoting histone exchange during transcriptional activation (23–25).

In the present study, we show that the CBP bromodomain interacts with the H3 K56 acetylation mark with a higher affinity than its previously characterized acetyl lysine-binding partners. In addition, we find that the CBP bromodomain interacts with the ASF1 histone chaperone via a nearby but distinct interface. From the structural perspective, a key disease mutation that we have identified in the CBP bromodomain can attenuate the interaction with both H3 K56Ac and ASF1. Functionally, we show that the bromodomain of CBP is critical for promoting the acetylation of free histones by CBP; this acetylation also is enhanced by ASF1. We find evidence that autoacetylation of CBP is critical not only for the substrate interaction with the HAT domain but also for the bromodomain to bind to its acetyl lysine-binding partners. These results clearly indicate that, although individual domains of CBP have designated functions, in the full-length context the protein conformation plays a key role in dictating its interactions and hence cellular function.

Results

The Bromodomain of CBP Shows Preferential Interaction with Acetylated H3 K56.

To investigate further the interaction between the CBP bromodomain and acetylated lysines within histones, a candidate-based approach was used. Escherichia coli-expressed recombinant GST-tagged CBP bromodomain was incubated with immobilized biotinylated acetylated peptides, and the relative amount of interacting GST-CBP bromodomain was measured by Western blotting against GST. In comparison with several acetylated histone peptides (H3 K9Ac, H3 K14Ac, H3 K18Ac, H3 K23Ac, H3 K27Ac, H3 K36Ac, H4 K5Ac, H4 K8Ac, H4 K12Ac, H4 K16Ac, and H4 K20Ac), H3 K56Ac showed the best binding with the bromodomain of CBP (Fig. 1A) (3). Among the well-studied interaction partners of the CBP bromodomain are H3 K36Ac, H4 K20Ac, and p53 K382Ac (5, 13). The interaction of the CBP bromodomain with H3 K56Ac appeared to be stronger, at least as measured by peptide pull-down assays, than the interaction with these previously characterized acetyl-binding partners (Fig. 1B). Because the bromodomains of CBP and p300 are highly similar (differing by only four amino acids), we asked whether the p300 bromodomain also recognizes H3 K56Ac. The CBP and p300 bromodomains showed efficient interactions with H3 K56Ac (Fig. 1C), whereas the less closely related human GCN5 bromodomain does not bind to H3 K56Ac (Fig. 1D). The binding affinity of the CBP bromodomain to the H3 K56Ac peptide subsequently was measured by isothermal titration calorimetry (ITC) and was found to be higher than that for the peptides previously identified as interacting with the CBP bromodomain (H3 K36Ac, H4 K20Ac, and p53 K382Ac (Fig. 1E). The binding of H3 K56Ac to the CBP bromodomain is an exothermic reaction, and the enthalpy is greater than in the interactions of the CBP domain with other known peptides.

Fig. 1.

Fig. 1.

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The CBP bromodomain binds to H3 K56Ac with higher affinity than to other monoacetylated lysines. (A) In vitro peptide pull-down interaction of the GST-CBP bromodomain with different site-specific acetylated histone peptides. (B) In vitro peptide pull-down interaction of the GST-CBP bromodomain with H3 K56 and H3 K56Ac peptides. (C) Comparison of the binding of the CBP and p300 bromodomains for H3 K56Ac and H3 K9Ac peptides. (D) Comparison of the H3 K56Ac-binding preferences of CBP and GCN5 bromodomains. (E) ITC shows that CBP bromodomain binds to the H3 K56Ac peptide but not to the unmodified peptide. H3 K36Ac, H4 K20Ac, and p53 K382Ac were used as positive controls. Kd values are shown.

To investigate whether the stronger interaction of the CBP bromodomain with H3 K56Ac, as compared with other acetyl lysine peptides, could be explained by additional contacts with the CBP bromodomain, we mapped the binding interface by NMR. H,15N heteronuclear single-quantum coherence (HSQC) spectra of the uniformly 15N-labeled CBP bromodomain were recorded while the H3 K56ac peptide (residues 49–59 of H3) or the corresponding unmodified H3 peptide was added gradually to the NMR samples. Substantial chemical shift changes in the bromodomain were observed during titration with H3 K56ac (Fig. 2A). A lack of resonance perturbations upon the addition of unmodified H3 peptide suggested that the CBP bromodomain does not recognize this peptide and that the acetylated Lys56 residue is essential (Fig. 2B). To characterize the H3 K56ac interaction structurally, we determined the crystal structure of the CBP bromodomain at a 1.4-Å resolution and defined the H3 K56ac-binding site by NMR (Fig. 2C). The overall fold of the CBP bromodomain in the apo state is similar to the previously determined fold of the protein bound to the H4 K20ac [Protein Data Bank (PDB) ID 2RNY] or p53 K382ac (PDB ID 1JSP) peptides (Fig. S1) (5, 13). The structures of the ligand-bound and unbound states superimpose over Cα atoms with rsmds of 1.5 Å and 2.4 Å, respectively, indicating that interaction with the acetylated peptides causes small conformational changes and that the binding site is essentially preformed.

Fig. 2.

Fig. 2.

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Structural analysis of the H3 K56Ac–CBP bromodomain interaction. (A and B) Superimposed 1H,15N HSQC spectra of the CBP bromodomain (0.2 mM) collected during titration with H3K56ac peptide (A) or unmodified H3K56 peptide (B) are color-coded according to the peptide concentration (Insets). (C) The histogram displays normalized 1H,15N chemical shift changes observed in the corresponding (A) spectra of the bromodomain. The red line indicates a significant change (greater than 0.1). The ZA and BC loops are indicated by green arrows. (D and E) The crystal structure of the CBP bromodomain. The protein is shown as a ribbon (D) or a solid surface (E). Residues that exhibit significant H3K56ac-induced perturbations in resonance are colored orange and are labeled. (F) Test of the effect of CBP bromodomain mutations on binding to immobilized H3 K56Ac peptide. The CBP bromodomain was GST tagged. (G) Test of the effect of CBP bromodomain mutations on binding to immobilized p53 K382Ac and H4 K20Ac.

The H3 K56Ac peptide binds to ZA and BC loops (Fig. 2 D and E), which are in the general area of the CBP bromodomain that mediates the interaction with other acetyl lysine-binding partners (5). However, several residues in this area were uniquely perturbed by H3 K56Ac, suggesting distinct contacts within the CBP–H3 K56Ac complex (Fig. S2). In the attempt to identify mutations that specifically disrupt binding to H3 K56Ac without compromising other acetyl lysine interactions of the CBP bromodomain, we generated D1116R, Q1118R, F1126A, and K1170E mutants. We found that although the residue substitution to an oppositely charged amino acid (in the case of D1116R) or the loss of the aromatic side chain (in the case of F1126A) reduces the interaction with H3 K56Ac (Fig. 2F), these mutations also disrupted the binding of p53 K382Ac and H4 K20Ac peptides (Fig. 2G). Furthermore, the Q1118R CBP bromodomain mutation, which had a marginal affect on the H3K56Ac interaction, greatly reduced binding to both p53 K382Ac and H4 K20Ac (Fig. 2G). These results are consistent with the idea that the weaker associations can be disrupted more readily than the interaction with H3 K56Ac (Fig. 1E).

Autoacetylation of CBP Is Required for the Bromodomain Interaction with Acetylated Lysines.

All previous analyses of the interactions between the CBP bromodomain and acetylated lysines had been performed only with the isolated CBP bromodomain. Therefore, we examined the ability of the CBP bromodomain to recognize acetylated lysines in the context of the full-length CBP protein. When we tested the ability of Sf9-expressed full-length CBP to bind to peptides carrying acetylated lysines, we found, to our surprise, that recombinant full-length CBP failed to bind to any of the acetylated peptides (Fig. 3A) to which the isolated CBP bromodomain bound (Fig. 1B). In comparison, full-length CBP that was expressed and purified from HeLa cells was highly effective in binding to both acetylated and to unacetylated histone peptides where the interaction with the acetylated peptide was mediated via the bromodomain and the interaction with the unacetylated peptide was mediated via the HAT domain (Fig. 3B). To investigate whether something in the HeLa cells rendered the full-length CBP capable of binding to acetylated and unacetylated histone peptides, we incubated the Sf9-expressed recombinant CBP with or without HeLa whole-cell extract. Strikingly, incubating the recombinant CBP with the HeLa cell extract fully restored its ability to bind to acetylated peptides via the bromodomain and to unacetylated histone substrates via the HAT domain (Fig. 3C). Because p300 is known to be autoacetylated, and this autoacetylation activates the HAT activity of p300 (12), we tested whether acetyl CoA was the component that was being supplied by the HeLa cell extract to activate the recombinant CBP so that it was able to bind not only to the substrates of the HAT domain but also to the acetyl lysine-binding partners of the bromodomain. Indeed, incubation of the recombinant full-length CBP with acetyl CoA was sufficient to render it able to bind to both the acetylated peptide and the unacetylated peptide (Fig. 3D). We confirmed that these conditions were sufficient to lead to autoacetylation of the recombinant CBP (Fig. 3E). We interpret these results as indicating that the unacetylated CBP has an inaccessible conformation in which neither the HAT domain nor bromodomain can recognize binding partners. We propose that autoacetylation of CBP promotes a conformational change that exposes the HAT and bromodomains so that these domains now can recognize their respective binding partners.

Fig. 3.

Fig. 3.

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CBP autoacetylation is required for the CBP bromodomain to bind to acetylated lysines. (A) Interaction of unmodified and acetylated H3 peptides with immobilized full-length recombinant CBP (CBP-FL). (B) Interaction of unmodified and H3 K56Ac peptides with FLAG-purified HeLa cells expressing FLAG-CBP. (C) Interaction of unmodified and H3 K56Ac peptides with immobilized baculoviral-expressed CBP upon incubation with HeLa whole-cell extract. (D) Interaction of unmodified and H3 K56Ac peptides with immobilized baculoviral-expressed CBP upon preincubation with acetyl CoA. (E) Autoacetylation of CBP can be detected by pan-acetyl antibody in the presence of acetyl CoA.

The Bromodomain of CBP Promotes Acetylation of Free Histones.

Next we sought to investigate the function of the interaction between the CBP bromodomain and H3 K56Ac. First, we asked whether the interaction is important for recruiting CBP to promoters that have increased levels of H3 K56Ac during transcriptional induction, such as the promoter of the Gene regulated in breast cancer 1 (GREB1) gene in MCF7 cells upon estradiol treatment (Fig. S3). We compared the ability of transfected FLAG-tagged full-length or bromodomain-deleted CBP to be recruited to the GREB1 promoter upon estradiol treatment by ChIP analysis and found that the two CBP constructs were recruited to the promoter with equal efficiency (Fig. S3). Therefore, the interaction between the bromodomain and acetylated histones, or any other protein for that matter, is not required for its recruitment to chromatin, at least at the GREB1 promoter.

Next we asked whether the interaction between H3 K56Ac and the CBP bromodomain can stimulate acetylation on free histones, given that CBP acetylates H3 K56Ac on free histones (18). We purified full-length CBP, CBP with the bromodomain deleted (∆bromo), or a catalytically inactive HAT mutant (HAT-ve) expressed from baculoviruses in Sf9 cells (Fig. 4A) (26). Using equal amounts of these CBP proteins in an in vitro HAT assay on recombinant H3/H4 tetramer and purified core histone substrates, we found that, unlike full-length CBP, ∆bromo CBP showed a compromised ability to acetylate H3 K56 in a time course-dependent manner (Fig. 4 A and B). These results suggested that the interaction between the CBP bromodomain and acetylated lysine promotes histone acetylation on free histones. To verify this result, we used a competitive inhibitor of the interaction between the bromodomain and acetylated lysines, called JQ1 (27). Upon titration of increasing amounts of JQ1 into the HAT assay with a constant amount of full-length CBP, we observed that acetylation of H3 K56 was inhibited in vitro in a concentration-dependent manner (Fig. 4C). This result indicates that the interaction between the bromodomain and acetylated lysines plays an important role in stimulating the acetylation of H3 K56Ac on free histones. To investigate whether the interaction of the bromodomain with acetylated lysine also was important for stimulating H3 K56 acetylation in vivo, we used the CBP/p300 bromodomain-specific inhibitor CBP-112 from SGC-Oxford and found that that it inhibited acetylation of H3 K56Ac but not of H3 K27Ac (Fig. 4D). Taken together, these results indicate that the CBP bromodomain stimulates acetylation of H3 K56 in a manner that is dependent on the interaction between acetylated lysine and the bromodomain.

Fig. 4.

Fig. 4.

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The CBP bromodomain stimulates acetylation of free histones. (A and B) In vitro HAT assays with recombinant H3–H4 (A) and purified core histones (B) with baculovirus-expressed full-length (FL), ∆bromo, or HAT-ve CBP. A Coomassie-stained gel of the purified CBP proteins is shown on the left. Acetylation is detected by Western blotting for H3 K56Ac. Amido black stains of the same membranes are shown below. (C) In vitro HAT assay with full-length CBP and increasing concentrations of the bromodomain interaction inhibitor JQ1 with core histones as substrate. Input H3 was assessed by Western blotting for unmodified H3 on the same membrane. (D) Alteration of H3 K56Ac but not of H3 K27Ac total levels upon treatment with increasing concentrations of CBP-112. Whole-cell extracts were prepared from HeLa cells treated with a range of CBP-112 concentrations (1, 2, and 5 μM) and were probed for α-H3 K56Ac and α-H3 K27Ac. (E) Comparative analysis of in vitro acetylation ability of equimolar amounts of full-length, ∆bromo, and HAT-ve CBP, the isolated HAT domain, and a combination of isolated HAT domain and isolated bromodomain. Amido black stains of the same membrane are shown below. A Coomassie-stained gel of the isolated CBP domains is shown on the right.

To distinguish between the possibilities that (i) the bromodomain further stimulates the endogenous activity of the HAT domain or (ii) the bromodomain overcomes the inhibitory effect of another domain on the HAT domain, we compared the HAT activity of equimolar amounts of full-length CBP, ∆bromo CBP, the isolated HAT domain, and the isolated HAT domain with the isolated bromodomain supplied in trans. Two interesting results were obtained from this experiment. First, the isolated HAT domain had higher HAT activity than full-length CBP (Fig. 4E). This result suggests that another domain of CBP inhibits the activity of the HAT domain, although it is possible that the recombinant HAT domain has a higher specific activity merely because of its mode of expression in E. coli. It is noteworthy that the RING domain of the related p300 protein recently has been shown to inhibit its HAT activity (28). Second, supplying the bromodomain in trans to the HAT domain did not further stimulate the HAT activity of the isolated CBP HAT domain, indicating that the CBP bromodomain does not enhance the endogenous activity of the HAT domain per se. From these results, we propose that the physical tethering of the bromodomain to the HAT domain somehow stimulates the catalytic activity of the HAT domain, perhaps through its ability to bind to acetylated proteins.

The Histone Chaperone ASF1 Binds to the Bromodomain of CBP.

We previously have shown that acetylation of H3 K56Ac in humans requires not only CBP but also the histone chaperone ASF1 (18). Furthermore, endogenous Drosophila ASF1 and CBP coimmunoprecipitate with each other (18). Humans have two isoforms of ASF1, ASF1A and ASF1B, which differ mainly in their C-terminal region. To investigate whether human ASF1A and/or human ASF1B interact with human CBP, we performed coimmunoprecipitation analyses from extracts made from HeLa cell lines stably expressing ASF1A-FLAG or ASF1B-FLAG. Both ASF1A and ASF1B coimmunoprecipitated with endogenous CBP (Fig. 5A). ASF1A previously has been shown to bind to the bromodomain of the CCG1 protein (29). Therefore, we wondered whether the bromodomain of CBP is important for the interaction with ASF1. To address this question, we immunoprecipitated FLAG-tagged full-length CBP, bromoΔ CBP, or HAT-ve CBP that was expressed following transient transfection into HeLa cells and determined how much ASF1A was coimmunoprecipitated. Compared with the binding of ASF1A to full-length CBP, the interaction with the bromoΔ CBP was greatly reduced, although the two constructs were expressed at similar levels (Fig. 5B). Given that the bromodomains of CBP and p300 differ by only four amino acids, it was not surprising to find that endogenous ASF1 also coimmunoprecipitated with p300 from HeLa cells (Fig. 5C). To determine whether ASF1 interacts directly with the bromodomain, we performed in vitro binding assays with ASF1A, ASF1B, or the conserved core of ASF1A (amino acids 1–167) and the bromodomain of CBP. All three versions of ASF1 interacted with the CBP bromodomain (Fig. 5D), indicating that the interaction is direct and is mediated by the conserved N-terminal region of ASF1. Furthermore, the bromodomain of p300 also binds to ASF1A (Fig. 5E). To determine which surface of ASF1 was contacting the CBP bromodomain, we performed NMR studies titrating the CBP bromodomain into H- 15N–labeled ASF1A (1–154). The residues of ASF1A that shifted upon interaction with CBP are indicated in Fig. 5F, and they all reside largely on one face of ASF1A. To confirm the contribution of these ASF1A amino acids to the interaction with the CBP bromodomain, we generated point mutations in ASF1A and showed that the I31R, T27R, E116R, and Y101F substitutions individually obliterated the interaction (Fig. 5G). The V45R mutation reduced the interaction, whereas mutation of L140 to an A or E reproducibly increased the interaction between the ASF1 and the CBP bromodomain. It is possible that these sidechains, which are shorter than the original leucine sidechain, either better fit the surface of the CBP bromodomain or are able to form better interactions. Regardless, the mutagenesis of ASF1 confirms that the residues identified by NMR indeed are important for the interaction with the CBP bromodomain. Interestingly, the interface of ASF1 that interacts with the CBP bromodomain is distinct from the interface that interacts with the CCG1 bromodomain (30).

Fig. 5.

Fig. 5.

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ASF1 binds to the bromodomain of CBP. (A) Coimmunoprecipitation of human ASF1A and ASF1B with CBP. Whole-cell extracts were made from stable cell lines expressing ASF1A-FLAG or ASF1B-FLAG and were tested for their ability to coimmunoprecipitate with α-CBP antibodies. The letter “p” denotes phosphorylated forms of ASF1. (B) Transient transfection of FLAG-tagged full-length, ∆bromo, and HAT-ve CBP in HeLa cells followed by M2-Agarose pulldown and probing for the presence of ASF1A in the complex by Western blot with an antibody specific to ASF1A. (C) Coimmunoprecipitation of ASF1A with p300 from HeLa cells. Whole-cell extracts were made from HeLa cells, and ASF1A was coimmunoprecipitated with α-p300 antibodies. (D) The CBP bromodomain binds directly to the N-terminal of ASF1A/B. The GST-CBP bromodomain was bound to ASF1A, ASF1B, or ASF1 (1–167) and analyzed by immunoblotting. (E) The p300 bromodomain binds directly to ASF1A. The recombinant GST-p300 bromodomain was bound to immobilized ASF1A and was analyzed by immunoblotting. (F) ASF1A interacts with the CBP bromodomain by NMR. The magnitude of the chemical shift perturbations for each amino acid of ASF1A, when CBP binds to it, and the location of these amino acids on ASF1 are shown. The SD of the chemical shift was 0.017 ppm. Residues with chemical shift changes greater than 2 SD are indicated in blue, changes greater than 3 SD in orange, and changes greater than 4 SD in red. (G) (Upper) ASF1A mutations showing compromised binding to the wild-type CBP bromodomain. (Lower) The purified ASF1 proteins used for the binding assay.

To determine whether the CBP bromodomain interacts with ASF1 via the same interface with which it interacts with acetylated histones, we performed NMR analysis titrations of ASF1A (1–154) with the H-15N–labeled bromodomain. The amino acids of the CBP bromodomain that showed chemical shift changes with the addition of ASF1 are shown in Fig. 6 A–C. Mutational analysis of the CBP bromodomain confirmed that CBP mutations K1180E, W1165A, N1183R, N1162R, and N1162E disrupt the ASF1–CBP bromodomain interaction (Fig. 6D). The mutant CBP bromodomain proteins were expressed and purified to equivalent levels (Fig. S4). Fig. 6E indicates the residues of the CBP bromodomain whose mutation disrupts the interaction with ASF1 or with the acetylated K56 peptide: ASF1 and H3 K56Ac bind to adjacent regions of the CBP bromodomain, although through different specific amino acids (Fig. 6E).

Fig. 6.

Fig. 6.

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Mapping the interaction of CBP with ASF1A. (A) HSQC spectra of the 15N-labeled CBP bromodomain in the absence (gray) and presence (red) of 1:1 ASF1A. The chemical shifts of CBP F1185 and R1163 are enlarged in the Inset. (B) Map of the chemical shift perturbations of the CBP interaction with ASF1. The changes in chemical shift induced in CBP by ASF1A are shown for each residue by number. The SD of the chemical shift was 0.07 parts ppm. Residues with chemical shift changes greater than 2 SD are indicated in blue, changes greater than 3 SD in orange, and changes greater than 4 SD in red. (C) The X-ray crystal structure of CBP mapping the ASF1A-binding site. Amino acids in CBP with chemical shift perturbations greater than 2 SD are shown on the crystal structure according to the color scheme in B. (D) Analysis of the effect of CBP bromodomain mutations on interaction with immobilized ASF1A. (E) Surface representation of the CBP bromodomain with ASF1-binding residues that were confirmed by mutagenesis in magenta and H3 K56Ac-binding residues that were confirmed by mutagenesis in marine blue. The Y1175 residue mutated in RTS is shown in green. (F) Cartoon representation of CBP bromodomain structure with Y1175 (present in wild type) marked in green and the corresponding RTS point mutant Y1175C marked in red. (G) Y1175C mutant CBP bromodomain shows compromised ASF1-binding ability.

A Mutation That Causes Rubinstein–Taybi Syndrome Disrupts the Interaction of the CBP Bromodomain with ASF1A and H3 K56Ac.

Upon superimposing the H3 K56Ac- and ASF1-binding sites onto the CBP bromodomain structure, we noted that both regions lay adjacent to the Y1175 amino acid (Fig. 6E) that is mutated to cause Rubinstein–Taybi syndrome (RTS) (31). Therefore, we introduced the Y1175 RTS mutation into the isolated CBP bromodomain and determined the effect on the interaction with ASF1A and H3 K56Ac. Mutating Y1175C in the CBP bromodomain did not affect the expression level or stability of the protein (Fig. S4) but did greatly reduce binding to the H3 K56Ac peptide (Fig. 2F) and to ASF1A (Fig. 6G). These results indicate that the inability of CBP to bind to acetylated histone H3 K56 and ASF1 could have direct implications in the pathogenesis of RTS.

The Bromodomain of CBP Is Critical for ASF1-Dependent Stimulation of the CBP HAT Activity.

Given that knockdown of ASF1A reduces CBP-mediated acetylation of H3 K56Ac in cells (18), we asked whether ASF1A also stimulates CBP-mediated acetylation in vitro. These assays were performed with limiting amounts of CBP and in conditions that favor the H3–H4 dimer, i.e., with the H3–H4 tetramer equilibrium tipped towards the H3–H4 dimer state. Furthermore, the histones were preincubated with ASF1A to promote formation of the ASF1A:H3–H4 dimer complex. We found that ASF1A enhanced acetylation of H3 K56Ac in a dose-dependent manner in vitro (Fig. 7A). However, ASF1A did not stimulate the HAT activity of CBP in general, because this same increase in acetylation was not observed for H2B K5Ac (Fig. 7A) or for p53 (Fig. S5).

Fig. 7.

Fig. 7.

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The interaction of ASF1 with histones and the CBP bromodomain stimulates histone acetylation. (A) HAT assays with either H3–H4 or H2A–H2B substrates in the presence of full-length CBP (CBP-FL) in the presence or absence of ASF1A. (B) (Upper) Transient transfection of full-length, ∆bromo, and HAT-ve CBP into HeLa cells, followed by analysis of the total H3 K56Ac and H3 K27Ac levels by Western blotting. (Lower) Quantification of acetylation. (C) Chromatin fractionation to monitor the distribution of H3 K56Ac between chromatin and free histones after transfecting CBP. Tubulin was used as the nonchromatin marker. (D) In vitro HAT assay to monitor acetylation mediated by full-length or ∆bromo CBP constructs in the presence or absence of ASF1. (E) HAT assay with CBP in the presence of wild-type or mutant (defective in interaction with bromodomain) ASF1. (F) HAT assay with CBP in the presence of increasing concentrations of wild-type or mutant (defective in interaction with histones) ASF1.

Overexpression of CBP is known to increase levels of acetylated histone substrates in cells above that mediated by the endogenous CBP (32). We also observed this increase with the acetylation of H3 K56Ac (Fig. 7B). Fractionation of the histones into chromatin-bound and non–chromatin-bound indicated that this additional H3 K56Ac that occurred upon overexpression of CBP resided not only on the chromatin-bound but also on the non–chromatin-bound histones (Fig. 7C), consistent with ASF1- and CBP-mediated acetylation occurring on free histones that later are incorporated into chromatin. Next, we examined whether the CBP bromodomain was required for the CBP-mediated acetylation of histones in cells. We found that overexpression of Δbromo CBP did not stimulate a further increase in H3 K56Ac levels over that mediated by endogenous CBP (Fig. 7B). However, the bromodomain was not required for the additional level of CBP-mediated acetylation of H3 K27Ac (Fig. 7B) or H3 K18Ac (Fig. S6). As such, this result shows that the CBP bromodomain is important for the ASF1-stimulated acetylation of H3 K56Ac by CBP in cells. To validate this result, we asked whether ASF1 can stimulate CBP-mediated acetylation of H3 K56Ac in the absence of the CBP bromodomain in vitro. Indeed, we found that ASF1 stimulated acetylation of H3 K56Ac on free histones in vitro by full-length CBP but not by Δbromo CBP (Fig. 7D). This result demonstrates that the bromodomain is required for ASF1 to stimulate CBP-mediated acetylation of free histones on H3 K56Ac.

Next we asked whether the interaction of ASF1A with the bromodomain or with histones is required for its ability to stimulate CBP-mediated H3 K56 acetylation on free histones. We found that the ASF1 I31R, T27R, and E116R mutations that disrupted the interaction between ASF1 and the CBP bromodomain (Fig. 5) had a greatly reduced ability to stimulate the HAT activity of CBP (Fig. 7E). This result demonstrates that the interaction between ASF1 and the CBP bromodomain enhances the ability of CBP to acetylate the histones. We also examined whether the interaction between ASF1A and histones is required for ASF1 to stimulate the CBP-mediated acetylation. The V94R mutation of ASF1A destroys its ability to bind to histones (33). This V94R mutant version of ASF1A did not stimulate CBP-mediated acetylation (Fig. 7F), indicating that the ASF1:H3–H4 interaction is critical for CBP-mediated acetylation of H3 K56 on free histones. Taken together, these data suggest that ASF1 physically recruits the histones to CBP for acetylation via the interaction between the CBP bromodomain and ASF1.

Discussion

In this study, we have uncovered two unique functions for the CBP bromodomain in promoting histone acetylation. First, the bromodomain promotes acetylation of free histones on H3 K56, presumably via the interaction between the acetylated lysine and the ZA and BC loops of the bromodomain. Furthermore, we have found that autoacetylation of CBP is required for the interaction between the bromodomain and acetylated histones to occur. Second, the bromodomain promotes the ASF1-stimulated acetylation of free histones on H3 K56, by interacting specifically with ASF1 via the side of the four-helix bundle of the bromodomain. We have characterized the interactions of the CBP bromodomain with H3 K56Ac and with ASF1A structurally and have identified mutations that disrupt these interactions. Finally, we show that a mutation in the bromodomain that causes RTS is defective in both of these interactions, suggesting that these interactions may be important for normal CBP function.

While our study was in progress, the Knapp laboratory reported that the CBP bromodomain also binds to H3 K56Ac with an affinity higher than that for its other previously published monoacetylated histone substrates (3). In their study, the affinities of the CBP bromodomain for all of the acetylated histone substrates were 5- to 10-fold higher than ours. However, it is important to note that interaction affinities measured by ITC or any other means are not absolute but instead are relative within an analysis that is performed under a fixed set of conditions. The relative affinities of the CBP bromodomain for the various acetylated histone substrates were similar in our study (Fig. 1E) and the previous work (3). We were unable to identify residues of the CBP bromodomain that uniquely mediate the interaction with H3 K56Ac but not other acetylated binding partners. This result suggests that the different acetylated binding partners bind to the same residues of the CBP bromodomain, but presumably the histone residues around K56 yield a more complementary fit with the surface of the CBP bromodomain than with the surface around the other acetylated lysines. As such, we are not able to disrupt only the interaction of H3 K56Ac with the CBP bromodomain to test the function of this interaction within cells. However, we did uncover an unexpected role for the CBP bromodomain in promoting acetylation on free histones.

A function for any bromodomain in promoting acetylation on non–chromatin-bound histones or proteins has not been demonstrated previously. Although it is possible that the bromodomain-acetyl K56 interaction may promote acetylation of the other H3 histone within the H3–H4 tetramer in vitro, this in unlikely to be the case in vivo given that most free H3–H4 exists bound to Asf1 as an H3–H4 heterodimer (34–36). Insight into the potential mechanism of the function of the CBP bromodomain in promoting acetylation can be obtained by looking at the related, much more highly studied protein p300. Although there is only 65% homology overall between CBP and p300, with each having unique physiological functions, their HAT domains are very highly conserved. The p300 HAT domain appears to function via a hit-and-run or Theorell Chance mechanism in which Acetyl CoA binds first, followed by the substrate (37). The rate-limiting step for the acetyl transferase activity of p300 has been proposed to be the release of the product (38). As such, one could imagine that the bromodomain could increase the rate of catalysis by binding to H3 K56Ac, thereby sequestering the product away from the active site of the HAT domain. Consistent with this notion, titration of competitive inhibitors of the acetyl lysine-binding pocket of the bromodomain blocked the ability of the bromodomain to stimulate acetylation of H3 K56 by the HAT domain (Fig. 4 C and D). This role for the bromodomain in promoting histone acetylation appears to be unique for promoting acetylation on free histones, because acetylation of H3 K27Ac on chromatin, which is mainly mediated by CBP and p300 (39), was not reduced by the CBP/p300-specific bromodomain inhibitor in cells (Fig. 4D).

Studies of the related p300 HAT domain have demonstrated that autoacetylation of an autoinhibitory loop is catalytically important for histone acetylation (12, 40). In this work we demonstrate that CBP also becomes autoacetylated. Furthermore, autoacetylation of CBP is required not only to allow the HAT domain to bind to unacetylated substrates but also to allow the bromodomain to bind to its acetylated binding partners. This result suggests that autoacetylation does more than enable the autoinhibitory loop and the inhibitory RING domain (28) to move away from the HAT active site. Functionally, the autoacetylation of p300 causes a conformational change within the p300 HAT domain that is detected by increased accessibility to proteinase K and promotes transcriptional activation (41). The autoacetylation of p300 also occurs beyond the autoacetylation loop, because p300 can be autoacetylated even when the autoacetylation loop is deleted (42). We suggest that, within the nonacetylated CBP, the acetyl-binding pocket of the bromodomain is blocked by another region of CBP. The CBP bromodomain does not bind to the HAT domain (28), indicating that the situation is more complicated than the HAT domain simply burying the acetyl-binding pocket of the bromodomain. This inaccessible conformation presumably is made more accessible upon CBP autoacetylation, enabling the HAT domain to bind to unacetylated substrates and the bromodomain to bind to acetylated products. It will be interesting to determine whether the cell uses the regulation of the autoacetylation state of CBP to control the interaction between the bromodomains and acetylated proteins.

The histone chaperone ASF1 has been shown previously to bind to bromodomains (29), but the mode of interaction with the CBP bromodomain is quite distinct from that observed with the CCG1 bromodomain (30). We find that the interaction between the CBP bromodomain and ASF1 and the interaction between ASF1 and histones promoted histone acetylation on free histones. We propose that, by interacting with the CBP bromodomain, histone-bound ASF1 forms a ternary complex in which H3 K56 is optimally presented to the HAT domain for acetylation by CBP. This model is reminiscent of the manner in which ASF1 promotes acetylation of H3 K56Ac on free histones by the yeast HAT Rtt109 (20, 23, 43). Our work shows that histone chaperones can also promote acetylation by a metazoan HAT enzyme, and we have uncovered the mechanism structurally. Furthermore, our studies extend the role of CBP/p300 from being HAT enzymes that acetylate chromatin-bound histones to also playing an important role in the acetylation of free histones in the cell.

CBP mutations are found in many human diseases, including many types of human tumors (44). Translocations involving CBP cause acute myeloid leukemia and mixed-lineage leukemia (44). RTS also is caused by mutations in a single copy of the CBP gene; it is reported that mutations leading to loss of HAT activity of CBP lead to RTS (45). These mutations also are present in the PHD finger of CBP that is critical for its acetylation activity (46). The RTS-causing mutation Y1175C lies immediately adjacent to the surfaces of the CBP bromodomain that we mapped by mutagenesis to mediate the interaction with ASF1 and H3 K56Ac. Interestingly, this CBP mutation implicated in RTS compromises the binding of both the acetylated histone and the histone chaperone. Our results suggest that the loss of H3 K56Ac and ASF1 binding to CBP may lead to the progression of RTS.

Materials and Methods

Site-Directed Mutagenesis and Protein Production.

The point mutants were generated using the Phusion SDM kit (New England Biolabs) following the standard protocol. Details of the expression and purification of the recombinant proteins are given in SI Materials and Methods.

Transient Transfection Assays.

Transient transfections of mammalian expression constructs of CBP (full-length, ∆bromo, and HAT-ve) were performed using Fugene HD following the standard protocol. A transfection time of 48 h was necessary for the expression of the transgene. M2-agarose pull down was performed, and then the complex was eluted from the beads with 0.1 M glycine HCl, pH 3.5. The solution subsequently was reequilibrated to neutral pH, diluted, and taken for further pull-down assays.

Peptide Pull-Down Assays.

One microgram of GST-tagged protein was incubated with 1 μg of biotinylated histone peptides in binding buffer [50 mM Tris⋅HCl (pH 7.5), 150 mM NaCl, 0.05% Nonidet P-40, 1mM DTT) overnight at 4 °C. Each binding reaction was incubated with Streptavidin Sepharose beads (Amersham) at 4 °C for 1 h. After binding the beads were spun down and washed with binding buffer three times at 4 °C. The beads subsequently were boiled with 5× SDS sample buffer and analyzed by Western blotting. Details of the peptides are provided in in SI Materials and Methods.

Immunoprecipitations.

FLAG immunoprecipitation was performed as described elsewhere (47). CBP and p300 immunoprecipitation were performed as described elsewhere (18).

Structural Analyses.

NMR and X-ray crystallography used standard techniques, as described in SI Materials and Methods.

HAT Assays.

HAT assays were performed following the standard protocol (48). Details are given in SI Materials and Methods.

Supplementary Material

Supporting Information
supp_111_12_E1072__index.html (7.2KB, html)

Acknowledgments

We thank Jay Bradner for the generous gift of JQ1; Danny Reinberg for the cell lines expressing FLAG-tagged ASF1A and ASF1B; Paul Lieberman for the kind gift of the baculovirus expression vectors and transient transfection vectors expressing full-length CBP, ∆bromo CBP, and HAT-ve CBP; Lee Kraus for the GST-p300 bromodomain; Peter E. Wright for the GST-GCN5 bromodomain constructs; Michelle Barton for purified recombinant p53 protein; Panagis Filippakopoulos for CBP-112; John Ladbury for generous assistance and advice with ITC and for assistance from the Center for Biomolecular Structure at the University of Texas MD Anderson Cancer Center; Jay C. Nix at Advanced Light Source Beamline 4.2.2; Catherine Musselman for helping with X-ray crystallographic and NMR data collection; and Jean Scorgie and Luke Smith for help with preparation of the ASF1 proteins. The use of core facilities was supported in part by University of Colorado Cancer Center Support Grant P30CA046934. This work was supported by NIH Grant GM64475 (to J.K.T.), a Cancer Prevention and Research Institute of Texas Rising Star and University of Texas Texas Stars and Senior Trust Recruitment Awards (to J.K.T.), and National Institutes of Health Grants R01GM079154 (to M.E.A.C.) and RO1GM096863 (to T.G.K.). C.D. was supported by a Susan Komen Race for the Cure Fellowship and a Ramalingaswami Fellowship from the Department of Biotechnology, Ministry of Science and Technology, Government of India.

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Data deposition: Atomic coordinates and structure factors reported in this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 4OUF and RCSB ID code rcsb084961).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1319122111/-/DCSupplemental.

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