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- Results and Discussion
The c-MYC oncoprotein regulates various aspects of cell behaviour by modulating gene expression. Here, we report the identification of the cAMP-response-element-binding protein (CBP) as a novel c-MYC binding partner. The two proteins interact both in vitro and in cells, and CBP binds to the carboxy-terminal region of c-MYC. Importantly, CBP, as well as p300, is associated with E-box-containing promoter regions of genes that are regulated by c-MYC. Furthermore, c-MYC and CBP/p300 function synergistically in the activation of reporter-gene constructs. Thus, CBP and p300 function as positive cofactors for c-MYC. In addition, c-MYC is acetylated in cells. This modification does not require MYC box II, suggesting that it is independent of TRRAP complexes. Instead, CBP acetylates c-MYC in vitro, and co-expression of CBP with c-MYC stimulates in vivo acetylation. Functionally, this results in a decrease in ubiquitination and stabilization of c-MYC proteins. Thus, CBP and p300 are novel functional binding partners of c-MYC.
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- Results and Discussion
The expression of genes is controlled by DNA-sequence-specific transcriptional regulators that recruit cofactors to modulate the activity of the polymerase complex (Narlikar et al., 2002). Cofactors function at least in part by affecting chromatin structure through their associated enzymatic activities, including ATPases/helicases, histone acetyl-transferases (HATs) and histone deacetylases (HDACs) (Jenuwein & Allis, 2001; Sudarsanam & Winston, 2000). Targets of the latter include histone tails that, depending on their modification status, function at least partly as interaction surfaces for proteins involved in the regulation of chromatin structure and gene transcription (Jenuwein & Allis, 2001).
MYC proteins, including c-MYC, N-MYC and L-MYC, are transcriptional regulators that were first identified as transforming factors. Subsequently, it was shown that MYC stimulates cell proliferation, inhibits differentiation and induces apoptosis in many cell types. Structure–function analyses have revealed several regions that are important for the ability of MYC to control cell behaviour. These include the amino-terminal transactivation domain (TAD) and the carboxy-terminal bHLHZip (basic helix–loop–helix zipper) domain, which is responsible for dimerization with its essential partner, MAX, and for sequence-specific DNA binding, respectively (Lüscher, 2001; Oster et al., 2002).
Little detailed information is available as to how c-MYC affects the activity of the polymerase II (Pol II) complex (Amati et al., 2001; Lüscher, 2001). Among the many proteins that have been shown to interact with c-MYC (Oster et al., 2002), TRRAP, a component of HAT-containing complexes, attracted particular interest (McMahon et al., 1998, 2000; Park et al., 2001). TRRAP binds to the MYC box II (MBII) motif in the N-terminal TAD of c-MYC, and is recruited to c-MYC-regulated promoters (Bouchard et al., 2001; Frank et al., 2001). The activation of some genes, including cyclin D2, telomerase reverse-transcriptase and nucleolin, by c-MYC requires MBII (Bouchard et al., 2001; Frank et al., 2001; Nikiforov et al., 2002). However, several other genes can be activated by c-MYCΔMBII, that is, in the absence of TRRAP recruitment (Nikiforov et al., 2002). These findings suggest that TRRAP is important for c-MYC-dependent regulation of some MYC target genes, whereas others may depend on additional MYC-associated activities.
c-MYC-dependent activation of target genes is associated with an increase in histone H3 and histone H4 acetylation (Bouchard et al., 2001; Frank et al., 2001). At least three distinct HATs, GCN5, PCAF and TIP60, can associate with TRRAP complexes. For two of these, GCN5 and TIP60, an interaction with c-MYC has been demonstrated (McMahon et al., 2000; B. Amati, personal communication). Thus, recruitment of these HATs to TRRAP-containing complexes is probably significant for gene regulation by c-MYC.
In this study, we identified the co-activator CREB (cAMP response element)-binding protein (CBP) as a novel c-MYC interaction partner. CBP and its close relative p300 are important transcriptional mediators for many transcription factors. CBP functions partly through its intrinsic HAT activity, as well as through associated HAT enzymes. In addition, CBP and p300 have been shown to interact with components of the Pol II complex, which may also contribute to co-activator function. CBP/p300 has several functions in the control of cell behaviour, including cell proliferation (Chan & La Thangue, 2001; Goodman & Smolik, 2000). Our findings show that CBP interacts directly with c-MYC and stimulates its function. Furthermore, CBP and p300 are recruited to MYC-regulated genes. Last, CBP acetylates c-MYC, thereby affecting its ubiquitination and half-life. Together, these findings identify CBP and p300 as novel functional binding partners of c-MYC.