In this report, we identified a novel cellular target for adenovirus DBP-induced host cell transcription control. We have found that DBP can bind to the SNF2-related CBP-activator protein, SrCap, and this interaction leads to inhibition of SrCap-mediated transcription. SrCap is a member of the SNF2 protein family that was cloned based on its ability to bind to CBP and was subsequently shown to function as an activator of CBP (13
). In addition, we have also found that SrCap activates transcription of several promoters, including the phosphoenolpyruvate carboxykinase (PEPCK), somatostatin, enkaphalin, and mouse mammary tumor virus promoters. This activation appears to function through CREB and glucocorticoid receptor-mediated transcription mechanisms (J. Chrivia, unpublished observations) and supports the model that SrCap functions as an activator of CBP.
In uninfected cells we demonstrate that SrCap and a series of cellular proteins are purified by coimmunoprecipitation using a SrCap-specific monoclonal antibody, indicating that SrCap is present in a multicomponent cellular protein complex. This is consistent with the fact that members of the SNF2 protein family exist in multiprotein complexes (11
). As expected, CBP and/or p300 proteins are present in SrCap-specific coimmunoprecipitations, consistent with the fact that SrCap was cloned as a CBP-binding protein and that members of the CBP/p300 protein family also interact with several cellular proteins, such as nuclear steroid receptors, basal transcription factors, and RNA polymerase II (8
It should be noted that recent advances in transcription and chromatin remodeling have led to the identification of two distinct families of chromatin remodeling proteins, those that remodel chromatin by targeting and modifying DNA structure in an ATP-dependent mechanism (i.e., SNF2 protein family) and those that remodel chromatin by targeting and modifying proteins that are essential for maintaining chromatin structure, through acetyltransferase activity (i.e., CBP/p300 protein family). It has been proposed that these families act in concert to coordinate remodeling of chromatin (22
). The SrCap-CBP/p300 association represents a cellular protein complex that contains members of both protein families. We speculate that the SrCap-CBP/p300 proteins may function synergistically in transcription and chromatin remodeling.
CBP/p300 proteins are key cellular targets of adenovirus growth control of host cells (19
). The adenovirus protein E1A has been demonstrated to block the transcriptional activity of several transcription factors which utilize CBP/p300 as a coactivator. E1A binds to at least four distinct regions within CBP/p300: the amino-terminal end, the histone acetyltransferase domain, and two sites in the C-terminal end. Binding of E1A to the amino-terminal end of CBP also blocks the binding of SrCap to this region (13
). This result suggested that adenovirus infection might alter the SrCap protein complex.
To our surprise, while E1A proteins were not observed, we found two adenovirus infection-specific proteins associated with the SrCap protein complex that migrated at 55 and 72 kDa. We have identified the 72-kDa SrCap-associated protein as the adenovirus 72-kDa DNA binding protein, DBP. We verified that the 72-kDa protein is DBP by using three separate experimental approaches: DBP was detected in a DBP-specific Western blot analysis of the SrCap complex, DBP was immunoprecipitated from denatured SrCap protein complex by DBP-specific monoclonal antibody, and the presence of DBP was indicated by comparing the partial proteolytic peptide digest pattern of 72-kDa SrCap-associate protein with that of DBP. The lack of a SrCap signal in the DBP-specific immunoprecipitation (Fig. , lane 5) is not surprising since only a small percentage of DBP is associated with a large cellular protein complex (24
The DBP-SrCap complex protein associations were not effected by micrococcal nuclease or ethidium bromide treatment, which digests or disrupts the structure of DNA, respectively. This indicates that although DBP is a DNA binding protein, association with the SrCap protein complex is not through nonspecific binding to DNA. These results are also consistent with previous studies that used these same techniques to demonstrate that DBP exists as part of a high-molecular-weight complex that is free of DNA (24
). In addition, our in vitro studies demonstrate that DBP interacts directly with SrCap. They indicate that a least one DBP binding site resides within the C-terminal end of SrCap.
Since the same carboxyl-terminal region of SrCap that was demonstrated to interact with DBP can function as a transcriptional activator when expressed as a Gal-SrCap fusion protein in a CAT reporter assay, we tested whether DBP could affect SrCap-mediated transcription. DBP was found to inhibit SrCap-mediated transcription in a dose-dependent manner, presumably through a direct protein-protein interaction with SrCap. The fact that there was no inhibition of the control Gal-VP16 transcriptional activator, even at the highest concentration of DBP, suggests that inhibition is specific for the presence of SrCap protein sequences in this system and argues against a model in which inhibition is due to a general nonspecific inhibitory effect resulting from DBP's DNA binding activity.
DBP-induced inhibition of a SrCap functional activity may contribute to the fact that DBP is toxic to cells, as demonstrated by the fact that stable DBP cell lines that constitutively express DBP cannot be made. In this model, DBP is inactivating the transcriptional activity and possibly the putative chromatin remodeling activity of SrCap. Since SrCap is an activator of CBP/p300 proteins, which are themselves chromatin remodeling proteins and key cell growth regulators that are implicated in regulating many transcription factors, a DBP-SrCap association could have detrimental effects on cell growth control.
Since the ability of adenovirus to mount a productive viral infection is highly species specific and DBP is implicated in host range determination, the DBP-SrCap interaction may also play a role in the determination of host range. This can be envisioned since mutations in DBP give it the ability to alter the host range of the virus (15
). These data make the DBP-SrCap interaction a potentially intriguing mechanism of host cell regulation by DBP.