MyoD, like other sequence-specific transcription factors, seems to be regulated by acetylation. The mechanism through which acetylation of MyoD increases its activity is not yet fully understood. MyoD acetylation by PCAF increases its affinity for DNA, at least for MyoD-MyoD homodimers (28
). However, homodimers are not the active species in muscle cells (19
), and thus the significance of this observation is not clear. Our results support an alternative hypothesis in which the acetylation of MyoD increases the stability of the complex formed between MyoD and the HAT CBP. This is concluded essentially from the analysis of acetylated MyoD in live cells and from results of in vitro testing. Such an effect of acetylation has been recently suggested for another transcription factor (29
). Concerning the mechanism of this stabilization, several alternative hypotheses must be considered. The acetylated lysines might be part of MyoD's domain of interaction with CBP, and the acetylation might facilitate this interaction, for example, through neutralization of repulsive charges. However, the region of MyoD in which the acetylated lysines are located has not been reported as being involved in the interaction with CBP or p300, for which a 100-aa N-terminal fragment seems to be sufficient (27
). Moreover, point mutation of the lysines did not impair the apparent affinity of nonacetylated MyoD for CBP in vitro (Fig. ). In addition, the interaction between CBP and acetylated MyoD did not require the C/H3 domain, the major domain of CBP for interaction with the N-terminal region of MyoD (Fig. ). As an alternative hypothesis, lysine acetylation could result in a conformational change of the MyoD molecule that would positively affect its interaction with CBP. Finally, the acetylated lysines themselves could provide a recognition motif for CBP (Fig. ). The last hypothesis is supported by the fact that the CBP bromodomain is involved in the interaction. In this regard it is significant that X-ray analysis of the PCAF bromodomain demonstrated that this domain selectively interacts with acetylated lysines (10
). A likely hypothesis is thus that the interaction between the CBP bromodomain and acetylated lysines in MyoD provides additional links that strengthen the interaction between the two proteins (Fig. ). In this model, the bromodomain would be a domain of protein-protein interaction that selectively recognizes amino acid sequences in which lysines are acetylated, as previously suggested (37
), much as the SH2 domains are domains that recognize amino acid sequences in which a tyrosine is phosphorylated. Our data provide the first experimental evidence for such a selective interaction between a nonhistone protein and the bromodomain: we show that the bromodomain of CBP, in the absence of the rest of the molecule, discriminates between acetylated and nonacetylated MyoD and selectively binds the acetylated but not the nonacetylated form. This result demonstrates that such a mechanism is not restricted to histones but rather may be more general.
A model for the association between acetylated MyoD and CBP (see Discussion). Br, bromodomain. Curved arrows, substrate acetylation by the HAT; asterisks, acetylated lysines on MyoD.
The interaction between recombinant MyoD and recombinant PCAF was weaker than that between MyoD and CBP and, in the absence of CBP, was not significantly strengthened by MyoD acetylation (27
; A. Polesskaya and A. Harel-Bellan, unpublished observations). However, it is likely that, in myogenic cells, PCAF is strongly associated with acetylated MyoD through CBP. Indeed, CBP-PCAF complexes are easily detected in muscle cells (A. Polesskaya and A. Harel-Bellan, unpublished observation), suggesting that significant proportions of the two molecules are associated in these cells. Moreover, the binding of MyoD and that of PCAF to CBP are not mutually exclusive, and a trimolecular complex could potentially exist (27
). The stabilization of the CBP-MyoD interaction thus likely facilitates the formation of a multimolecular complex that includes PCAF.
Mutation of acetylation sites in MyoD strongly decreased the ability of the molecule to functionally cooperate with CBP (Fig. ). The mutant, however, was at least partly rescued by a large excess of CBP, suggesting that its defect resides in the lack of a strong interaction with CBP and the HAT complexes. A strong interaction between MyoD and the HATs might have several consequences. Given that sequence-specific transcription factors have been shown to recruit HAT complexes to target promoters (34
), a strong interaction between CBP and acetylated MyoD is likely to result in a more efficient recruitment of the HATs to muscle-specific promoters. In that regard, it should be noted that CBP and p300, which are central to the activity of a wide variety of transcription factors, are thought to be present in limiting amounts in cells. Competition of transcription factors for these pivotal coactivators could affect gene regulation (15
). In support of this hypothesis is the observation that cbp
is subjected to major gene dosage effects in mice (41
) and in humans (23
). A strong interaction between MyoD and the HATs could thus help in maintaining an adequate fraction of the HATs complexed to MyoD in muscle cells. In this regard, it should be noted that the muscle differentiation program is irreversible, and a strong interaction between MyoD and the HATs could play a role in this phenomenon. The HAT-MyoD complex is likely to be strongly bound to muscle-specific promoters such as MCK, where a significant increase in the acetylation of histone H4 is observed during terminal differentiation of myoblasts (Fig. ). Acetylation of core histones has been reported to have a very short half-life (14
), and permanent reacetylation of histones is likely to be necessary to maintain an “open” structure on the promoter. A strong association between acetylated MyoD and CBP or p300 might thus be crucial to retaining the HAT complex on muscle promoters and maintaining, locally, a state of hyperacetylation.
In summary, our results lead us to propose a model in which MyoD acetylation allows the sequestration of HAT complexes to muscle-specific promoters on which they acetylate other substrates such as histones. These data reveal a new mechanism for transcription factor activation by acetylation. In addition, they provide the first experimental evidence for a selective interaction between an acetylated nonhistone protein and a bromodomain, leading to the generalization of a hypothesis previously formulated for the function of these domains.