The transactivation domain of the papillomavirus E2 protein is crucial for interaction with the Brd4 protein; proteins lacking this domain do not interact with Brd4, and the R37-I73 mutation disrupts binding (5
). However, we demonstrate here that the isolated transactivation domain has much weaker affinity for Brd4 than the full-length protein. This finding seems at odds with the results of previous studies in which we demonstrated that the E2 transactivation domain is sufficient for interaction with mitotic chromosomes (4
). However, in those studies, the domain was found to be associated with mitotic chromosomes in only a low percentage of cells, which we concluded was due to poor protein expression.
In this study, further analysis of the E2-Brd4 interaction demonstrated that neither the hinge region of E2 nor the DNA binding function is required for Brd4 interaction but that the dimerization function of the C-terminal domain is necessary for strong binding. Both the in vivo chromosomal binding and in vitro Brd4 binding role of the dimeric DNA binding domain of E2 can be fulfilled by the corresponding dimeric DNA binding domain of Gal4 or EBNA-1, but not by the monomeric RFP. Efficient Brd4 binding, both in vivo and in vitro, could also be retained by the fusion of E2 to an FKBP12-derived domain in which dimerization could be regulated by a small-molecule ligand.
In further support of the finding that E2 dimerization is important for efficient Brd4 binding, Kurg et al. generated a novel E2 single-chain heterodimer protein which contained one transactivation domain linked to a dimeric DNA binding domain (16
). This protein can bind Brd4, but much less efficiently than the wild-type E2 protein. It can also activate transcription in a Brd4-dependent manner, but the requirement for Brd4 is also much less than that of the wild-type protein.
The E2 transactivation domain has also been implicated in self-interaction, and the looping of DNA containing E2 binding sites has been observed previously by electron microscopy for BPV-1, human papillomavirus type 16 (HPV-16), and HPV-11 E2 proteins (3
). For HPV-16 E2, self-interaction involves residues R37 and I73, which are also crucial for Brd4 binding (1
), and thus, self-interaction may modulate Brd4 binding. However, the precise interactions between N-terminal domains of different E2 proteins are not consistent and may result from nonspecific crystal packaging (14
). A structure of the BPV-1 N-terminal domain revealed a disulfide bond between cysteine residues at positions 57 in the two monomers, which interfered with the E1 interaction face of the domain (25
). A different interface was also observed previously in the protein crystal lattice obtained for the HPV-11 E2 protein (31
). Thus, while self-interaction through the N-terminal domain may be important for E2 function, more studies are required to determine the precise details for each E2 protein.
We propose that the dimerization of E2 through the C-terminal domain enables two transactivation domains to interact with two copies of the Brd4 protein. This pattern would increase the local concentration of E2 interaction surfaces and thereby increase Brd4 binding. In support of an E2-Brd4 heterotetramer, a recent crystallographic structure of the HPV-16 E2 transactivation domain in a complex with a C-terminal 20-amino-acid peptide from Brd4 demonstrated that two Brd4 peptides were found sandwiched between two E2 N-terminal domains (1
). The dimerization of the E2 protein through the C-terminal domain would greatly stabilize such a tetramer complex. It has also recently been shown that Brd2, a protein related to Brd4, forms dimers mediated through one of the bromodomains (bromodomain 1) (22
). If Brd4 were to dimerize in a similar fashion, E2 and Brd4 could also form higher-order complexes whereby each dimeric E2 protein could interact with the C-terminal tails from two different dimeric Brd4 proteins. This cooperative binding would increase the local binding of E2 and Brd4 and potentially allow the formation of large complexes consisting of a lattice of multiple E2 and Brd4 dimers, which could explain why the Brd4-E2 complex is observed as punctate speckles on mitotic chromosomes (21
) whereas, in the absence of E2, Brd4 forms a diffuse coating (10