Activation of Wnt target genes by β-catenin/Tcf is tightly controlled by a complex network of regulatory events, including interactions with various cellular partners and different covalent modifications of β-catenin and Tcf4 (17
). Notably, the role of CBP and p300 as coactivators of β-catenin is well established, and their recruitment to Tcf-dependent promoters plays a crucial role during development and cell transformation (20
). In this study, we have shown that β-catenin is acetylated by p300 in vivo and in vitro and identified β-catenin residue K345 as a target site for acetylation. Substitution of K345 greatly reduced but did not totally eliminate the acetylation signal in vivo. These results are consistent with a recent report showing that CBP acetylates residue K49, located in the N-terminal domain of β-catenin (50
). In both studies, PCAF was unable to acetylate β-catenin, indicating that β-catenin acetylation is specifically achieved by CBP/p300. Whether acetylation modulates β-catenin functions is therefore an important issue.
K345 is located in arm repeat 6, a region implicated in β-catenin interaction with a variety of cellular partners. Our mutagenesis studies indicate that K345 is not significantly involved in β-catenin interaction with APC or Axin 1, but it was found to be critical for Tcf4 binding. Interestingly, mutation to alanine abolished the interaction with Tcf4 but mutation to arginine had no effect, probably because the positively charged arginine can maintain the interaction with the negatively charged residues of Tcf4. This interpretation is supported by a recent structural analysis of the β-catenin/Tcf complex, suggesting that charged residues around β-catenin K312, including K345, might interact with the negatively charged region of Tcf4 extending from E23 to E29 and facilitate the initial anchorage of Tcf4 to β-catenin (13
). The finding that the β-catenin mutant K345R was still able to bind Tcf4 suggests that acetylation does not serve merely to neutralize a positive charge but may create a novel interface facilitating the binding of β-catenin to Tcf4. Similarly, acetylation has been shown to increase the DNA binding potential of E2F1, although arginine mutants retained the ability to bind DNA (31
). The role of K345 in the β-catenin-Tcf4 interaction was further confirmed by the finding that the capacity of the K345A and K345R mutants to bind Tcf4 was strictly correlated with their ability to transactivate the TOPFLASH reporter (Fig. ). Reduced transactivation efficiency has also been demonstrated previously for a series of β-catenin mutants defective in Lef-1 binding (46
). This effect was not apparently related to abnormal subcellular localization of these β-catenin mutants, since exogenously overexpressed mutants accumulated in the nucleus at the same rate as β-catenin (data not shown). It is also unlikely that substitution of lysine 345 might affect the stability of β-catenin, since it does not impair β-catenin interaction with APC and Axin 1, two major partners within the degradation complex.
An important aspect of our results is that K345 acetylation increases the affinity of β-catenin for Tcf4. This conclusion is supported by several lines of evidence (see Fig. to ). (i) In the EMSA, p300 increased the binding of the wild-type β-catenin arm domain (arm 1-12) to Tcf4 but had no effect on the corresponding K345R mutant. (ii) In competition assays, a peptide containing acetylated K345 (ac-arm 6 peptide) disturbed this interaction in a dose-dependent manner, more efficiently than the homologous nonacetylated peptide, and (iii) comparable results were also obtained in GST pull-down experiments. (iv) In coimmunoprecipitation assays, the acetylated fraction of endogenous β-catenin was found to associate preferentially with Tcf4. Moreover, the increased affinity of acetylated β-catenin for Tcf4 affected the transcriptional activity of the complex, since cooperation between p300 and β-catenin was significantly reduced upon alteration of lysine 345 of β-catenin to arginine, although this mutant was still able to bind Tcf4. However, the transactivating activity of β-catenin/Tcf complexes involves the recruitment of a large number of cofactors through the β-catenin arm domain (17
). Therefore, we cannot completely rule out that acetylation of K345 could also influence the binding of such factors and thereby participate in transcription regulation.
Importantly, we observed that HAT-deficient p300 had little effect on β-catenin-mediated activation of the TOPFLASH reporter (Fig. ) and the natural β-catenin-responsive interleukin-8 promoter (data not shown), implying that the acetyltransferase activity of p300 was required for its coactivator function. In addition, while p300 retained some capacity to coactivate the K345R β-catenin mutant, the HAT-deficient p300 was unable to coactivate this mutant. These results strongly suggest that recruitment of p300 by β-catenin on Tcf-dependent promoters fulfills different functions, as previously described in different settings (26
). Besides its ability to bridge DNA-associated activators to the basal transcription machinery, p300 might act by altering chromatin structure though intrinsic HAT activity and modulate β-catenin activity though its factor acetyltransferase activity. Such functional duality has been convincingly demonstrated for coactivation of HMGI(Y) and p65 by CBP/p300 or PCAF, because in these cases, the HAT activity stimulates transcription, but the factor acetyltransferase activity could serve the opposite function in turning off transcription (21
). Alternatively, as proposed by Miller and Moon (32
), overexpressed mutant β-catenin could compete with endogenous β-catenin for binding to the degradation complex, leading to nuclear accumulation of both wild-type and mutant proteins.
Although coactivation of β-catenin by p300 is well established (20
), the contribution of HAT and factor acetyltransferase activities has received little attention so far. Recently, Hecht and collaborators reported that coactivation of β-catenin at the siamois
promoter is independent of the acetyltransferase activity of CBP/p300 (20
). The reasons for the apparent discrepancy between this observation and our present data are unclear, but it might be explained by recent results showing that different Tcf proteins such as Lef1 and Tcf4E could perform specific, nonredundant functions at different natural β-catenin-responsive promoters (19
). It will be of interest to determine whether the acetyltransferase activity of p300 is needed for coactivation of β-catenin in different promoter contexts and whether acetylation of β-catenin affects its binding to different members of the Tcf family. Although we have observed already that acetylation of K345 but not that of K49 mediates, at least in part, the transcriptional coactivation of β-catenin/Tcf by p300, further research on the functional effect of lysine 49 acetylation could determine whether it is implicated in modulating the acetylation of lysine 345.
Besides their role in Tcf-dependent transcription, it has been shown that β-catenin and CBP/p300 are transcriptional coactivators of the AR. Chesire and Isaacs (10
) put forward the notion of a reciprocal balance between the activation of AR- and Tcf-related transcription by β-catenin, which might be involved in normal prostate development and in prostate tumor progression. In this study, we showed that acetylation of β-catenin at K345 specifically increases the affinity of β-catenin for Tcf4 but seems to decrease the affinity of β-catenin for the AR (Fig. ). One might thus speculate that this posttranslational modification of β-catenin is involved in differential gene activation through Tcf versus the AR.
In conclusion, the role of p300 in the regulation of Wnt signaling appears to be complex. Thus, acetylation might participate in the activation of specific sets of genes upon Wnt signaling.