In the present study, we have attempted to identify the factors in striated muscle that interact with Rb. We have cloned a novel protein, EID-1, expressed in cardiac and skeletal muscle that specifically interacts with Rb through a LXCXE motif located in its C terminus. Developmental expression patterns and overexpression studies of EID-1 suggested that this molecule represented a novel inhibitor of differentiation. While in vitro binding studies and two-hybrid assays strongly support the notion that EID-1 interacts with Rb, we have been unable to directly show an interaction of the endogenous proteins. This may be simply related to technical limitations of our reagents or the low levels of expression of the two factors. Despite this, our data suggest, at least indirectly, that the interaction of EID-1 with Rb is functionally important. The point mutation in EID-1 that abolishes Rb binding was a more potent inhibitor of MyoD-dependent transcription. Therefore, our data support an alternative model for the interplay of Rb and MyoD during skeletal muscle cell differentiation that would be dependent on the expression of Rb but does not require a direct Rb-MyoD physical association. In support of this premise, data are presented in the accompanying manuscript by Miyake et al. that not only can Rb rescue EID-1's inhibitory effects but that overexpression of EID-1 can disrupt some aspects of Rb function (42
). EID-1 joins a growing list of inhibitors of differentiation, including p202 and HBP1, that have been shown to interact with Rb (10
). The fact that several inhibitory factors have been identified which interact with Rb suggests that this protein may represent a differentiation checkpoint, linking factors regulating cell cycle exit and tissue-specific gene expression (60
Interestingly, Rb has also recently been shown to be critical for MEF2-dependent transcriptional activity (45
). This potentiation of MEF2 activity was in part independent of Rb's effects on cell cycle progression, but the basis of this cell cycle-independent effect was not determined. Since MEF2 transcriptional activity (55
), like that of MyoD, has been reported to be p300 dependent, an indirect mechanism involving EID-1 might explain both the defect in skeletal muscle gene expression in the absence of pocket proteins (19
) and our and others' inability to confirm a physical interaction between Rb and MyoD in vivo (45
). Additionally, it might explain the paradoxical observation that Rb can potentiate the transcriptional activity of certain factors (8
) despite its inherent transcriptional repressor-like activity (59
). This link between Rb and p300 provides a cogent hypothesis to explain these discordant results.
The similarities between EID-1 and E1A are obvious; however, several differences are also apparent. The most obvious is EID-1's lack of effect on cell cycle progression. A priori, based on results with classic LXCXE proteins, E1A and SV40 large T Ag, one would have predicted cell cycle reentry as the default hypothesis; however, there are now multiple reports that endogenous cellular proteins with LXCXE motifs can have divergent effects on the cell cycle (29
). This may in part be explained by the ability of pocket proteins to bind multiple partners simultaneously (61
). A model for this regulatory activity has been proposed whereby Rb effects on cell cycle are separable from its differentiation-promoting properties (58
): Rb-dependent growth arrest requires an intact E2F binding site, while E2F binding was dispensable for Rb to promote differentiation, suggesting that Rb was binding and modulating a second, functionally distinct class of proteins.
HATs play a critical role in tissue-specific transcription by relieving repressive effects of chromatin condensation. p300 is a structural and functional homologue of CREB-binding protein, a transcriptional coactivator that not only has intrinsic HAT activity (46
) but is capable of recruiting additional HAT factors to the transcriptional complex (76
). p300, which was originally cloned as an E1A-binding protein, was subsequently shown to be critical for normal skeletal muscle differentiation, since disruption of its function by neutralizing antibodies or dominant-negative mutations blocks both differentiation and cell cycle arrest (49
). E1A mutations that selectively block p300 function are capable of inhibiting skeletal and cardiac muscle differentiation (32
). Until recently, it was presumed that competitive binding was the basis for the ability of E1A to inhibit tissue-restricted expression in skeletal muscle, since E1A interacts with the same region of p300 as MyoD (C-H3 domain). However, E1A has now been shown to directly inhibit the HAT domain of p300 or of the p300- and/or CBP-associated factor, PCAF (6
). We have provisionally suggested an analogous mechanism for EID-1, whose interaction with p300 in two-hybrid assays was specifically contingent on the C-H3 domain (Fig. C) and could inhibit p300 HAT activity (Fig. ). Whether this inhibition of HAT activity is a general model for differentiation inhibitors will need to be determined; however, Twist, a skeletal muscle inhibitor, has already been shown to interact with p300 and inhibit its HAT activity (23
In summary, our data suggest a novel mechanism for the interplay of Rb and MyoD and for the dependence of skeletal muscle cell differentiation on p300 and/or CBP. Further studies of EID-1 are in progress to delineate whether EID-1 also directly effects MyoD, possibly by inhibiting PCAF-dependent acetylation which has recently been shown to be important for MyoD DNA binding and transcriptional activity (56
). Although EID-1 appears to be highly expressed in striated muscle and brain tissue, its expression in adult tissues is widespread, albeit at lower levels. Since Rb has been postulated to play a role in the differentiation of a wide variety of tissues, EID-1 and the model proposed may represent a more generalized mechanism of differentiation regulation. The extent of this role will await studies detailing its developmental expression and the creation of a mouse model deficient in this protein.