Transcription coactivators (reviewed in reference 45
) can function not only to transmit the signal of ligand-induced conformational change to the basal transcription machinery but also to modulate chromatin structure. In this report, we have described the initial characterization of ASC-1, which shows various properties consistent with its role as a novel transcription coactivator molecule of nuclear receptors. These include associations with nuclear receptors (Fig. and ), the basal transcription machinery (Fig. ), and transcription integrators CBP and SRC-1 (Fig. ), an autonomous transactivation function (Fig. ), and coactivation of transactivation mediated by nuclear receptors (Fig. ).
The interactions of ASC-1 with nuclear receptors are ligand independent in vitro (Fig. B), whereas inclusion of SMRT (20
) in the binding reactions resulted in ligand-dependent interactions of ASC-1 with receptors (Fig. C). The hinge domain of nuclear receptor is a major determinant in interactions with ASC-1 (Fig. A) as well as SMRT (20
). Since SMRT is released from liganded nuclear receptors and ASC-1 is still able to bind to liganded receptors, the receptor–ASC-1 bindings should become ligand dependent in vivo, where SMRT is ubiquitously expressed. The indirect immunofluorescence results are also consistent with this notion (Fig. ). However, it was intriguing that the relatively strong basal interactions of ASC-1 with receptors were further stimulated by ligand in yeast, whereas the interactions of ASC-1 with TR-DEF459, a previously described AF2 mutant TR (29
), were not. These results may have been caused simply by ligand-induced stabilization of receptor conformation to facilitate the ASC-1–receptor interactions in yeast. Alternatively, proteins functionally homologous to SMRT, an AF2-dependent coactivator such as SRC-1–CBP-p300, or both may exist in yeast, and ASC-1 may cooperate with these putative yeast proteins to mediate the ligand- and AF2-dependent coactivation of nuclear receptors in yeast. In this regard, it is notable that our database search revealed the highly conserved ASC-1 homologues in the higher eukaryotes Caenorhabditis elegans
and Schizosaccharomyces pombe
as well as a relatively distant ASC-1 homologue in S. cerevisiae
(results not shown).
Recent biochemical studies suggest that transcription coactivators function as a complex with other related proteins (45
). Thus far, several groups of such distinct macromolecular complexes have been described. The TAF components of TFIID (reviewed in reference 4
) and the SRB-MED components bound to polymerase II (reviewed in reference 38
) comprise those that are ultimately associated with the general transcription machinery. B-cell-specific OCA-B (35
), a group of distinct nuclear proteins named thyroid hormone receptor associated proteins (TRAPs) (12
), and a transcriptionally active nuclear complex that interacts only with liganded vitamin D receptor (VDR) (DRIPs) (43
) define a group of coactivator complexes with rather specific functions. TRAPs purified from HeLa cells grown in the presence of thyroid hormone (T3) were found to markedly activate transcription by liganded TR in vitro, whereas DRIPs consisting of a complex of at least 10 different proteins ranging from 65 to 250 kDa were found to coactivate the VDR-dependent transactivation. The DRIPs were distinct from the CBP-p300 complex, although like these coactivators, their interaction also required the AF2 transactivation motif of VDR (43
). Finally, the CBP-p300 coactivator complex defines a distinct coactivator complex that directly binds and coactivates a wide spectrum of different transcription factors. In particular, CBP-p300 was found to be complexed with SRC-1 (18
) and a series of cellular proteins with molecular masses ranging from 44 to 270 kDa (11
). Purification and analysis of various proteins in this group revealed that they are components of the human SWI-SNF complex and that p270 is an integral member of this complex. In addition, different classes of mammalian transcription factors—nuclear receptors, CREB, and STATs—were recently shown to functionally require distinct components of the CBP-p300 coactivator complex, based on their platform or assembly properties (27
). RAR, CREB, and STATs were further demonstrated to require different histone acetyltransferase activities within the CBP-p300 complex to activate transcription. Recently, p300 and CBP, despite their similarities, have been shown to have distinct functions during retinoid-induced differentiation of embryonic carcinoma F9 cells (24
). Overall, these results suggest that distinct coactivator complexes appear to exist among CBP-p300-containing coactivator complexes in the cell, which should be responsive to distinct activating signals and differentially integrate various signaling pathways.
We have presented a few pieces of experimental evidence that support direct associations of ASC-1 with SRC-1 and CBP-p300. First, ASC-1 was shown to physically bind CBP-p300 and SRC-1, as demonstrated by the yeast two-hybrid tests and the GST pull-down assays (Fig. ). Second, ASC-1 was found to colocalize, at least under serum-starved conditions, with CBP and SRC-1 in vivo, as demonstrated by the indirect immunofluorescence of Rat-1 fibroblast cells (Fig. ). Finally, while the ASC-1 interaction domain does not include the AF-2 domain of nuclear receptors (Fig. ), ASC-1 was shown to cooperate with SRC-1 and p300 in coactivating transactivation by nuclear receptors (Fig. A). In addition, ASC-1 was not able to coactivate the mutant RXR that lacks the AF2 domain (i.e., RXRΔAF2) (Fig. B), clearly indicating that ASC-1 has to function in conjunction with AF2-dependent factors such as CBP-p300 and SRC-1. These results suggest that ASC-1 may represent an active member of the CBP-p300 complexes, along with SRC-1 and related proteins. Alternatively, it may represent a constituent of distinct coactivator complexes that, in turn, functionally interact with the CBP–SRC-1 complexes. Consistent with the latter possibility, it was recently shown that CBP, p300, and SRC-1 exist as distinct steady-state coactivator complexes in vivo (37
). In addition, we have also isolated a novel complex of proteins from HeLa nuclei based on their tight association with ASC-1 which exhibited a fractionation profile distinct from that of CBP, p300, or SRC-1 and didn’t appear to contain any of these proteins (unpublished results).
One of the most striking features of ASC-1 was its interesting relocation property (Fig. ). When deprived of serum, ASC-1 accumulated in cytoplasm, while it was exclusively nuclear in the presence of ligand or coexpressed CBP or SRC-1. However, it is not known whether this cytoplasmic accumulation under conditions of serum deprivation is due to the inability of newly synthesized ASC-1 to enter the nucleus or active relocation of the existing nuclear ASC-1 to the cytoplasm. Nonetheless, these relocation properties raise an interesting possibility that ASC-1 may play a critical role in establishing distinct coactivator complexes under different cellular conditions (as summarized in Fig. ). When deprived of serum, for instance, coactivator complexes devoid of ASC-1 may predominate within the nucleus (due to the cytoplasmic accumulation of ASC-1), which may preferentially transactivate a set of transcription factors activated by serum starvation while shutting down general transcription activities. Candidate genes potentially regulated by these non-ASC-1-containing coactivator complexes include those specifically expressed at growth arrest (10
). It is also notable that ASC-1 is likely to function with transcription factors other than nuclear receptors, based on its associations with SRC-1 and CBP, which in turn functionally interact with transcription factors in diverse signaling pathways. Indeed, we have found that ASC-1 is required for transactivation by multiple transcription factors, including AP-1 and NF-κB (unpublished data).
FIG. 10 Model of ASC-1 actions. Distinct putative coactivator complexes, each containing ASC-1, SRC-1, or CBP, exist in vivo (37 and unpublished results) and mediate transactivations by a number of distinct transcription factors. Each of these complexes recognizes (more ...)
In conclusion, we have described a novel coactivator molecule capable of associating with liganded receptors, SRC-1, and CBP in vivo and which may play a critical role in integrating different cellular conditions into the transcription machinery. Surprisingly, all of the functions of ASC-1 described in this report appear to require only the putative zinc-binding domain, whereas the actions of the much larger CBP, p300, and SRC-1 proteins are dependent on distinct interaction domains for their various targets. However, it should be noted that only some of these interactions are likely to exist within the ASC-1-containing complex. Finally, it’s notable that ASC-1 contains multiple phosphorylation sites, potentially responsive to various signals, suggesting that ASC-1 may directly respond to various cellular regulatory signals. Overall, studies of this coactivator protein should provide important insights into the multifactorial control of biological processes under regulation of multiple signal transduction pathways in vivo.