Brd4 Is Involved in Multiple Processes of the Bovi
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病菌学杂志 2006年第7期
Institute of Technology
Department of Microbiology and Virology, Institute of Molecular and Cell Biology, University of Tartu and Estonian Biocentre, Tartu, Estonia
ABSTRACT
Brd4 protein has been proposed to act as a cellular receptor for the bovine papillomavirus type 1 (BPV1) E2 protein in the E2-mediated chromosome attachment and mitotic segregation of viral genomes. Here, we provide data that show the involvement of Brd4 in multiple early functions of the BPV1 life cycle, suggest a Brd4-dependent mechanism for E2-dependent transcription activation, and indicate the role of Brd4 in papillomavirus and polyomavirus replication as well as cell-specific utilization of Brd4-linked features in BPV1 DNA replication. Our data also show the potential therapeutic value of the disruption of the E2-Brd4 interaction for the development of antiviral drugs.
TEXT
Papillomavirus (PV) E2 protein is a central regulator of the viral life cycle. In addition to its well-established activity as a transcription modulator and replication initiator protein (6), E2 of bovine papillomavirus type 1 (BPV1) has recently emerged as a trans factor which mediates mitotic segregation of viral genomes by tethering them to host cell chromatin (7, 12, 19). The first candidate for a receptor of E2 in the latter process, Brd4, is attached to the chromatin through its two bromodomains, which bind to acetylated histones H3 and H4 both in interphase and in mitosis (4, 25). Mutated E2 proteins that are defective in Brd4 binding are unable to bind to mitotic chromosomes (2), and ectopic expression of Brd4 can reconstitute the BPV1 E2-dependent extrachromosomal plasmid maintenance in the yeast Saccharomyces cerevisiae, where such a process normally does not function (3). Ectopic expression of the E2-binding C-terminal domain (CTD) of Brd4 in mammalian cells disrupts the interaction of E2 with cellular Brd4 and relocates E2 from mitotic chromosomes (25, 26). Brd4 CTD binds to the N-terminal domain of E2 (25), which is also responsible for interactions critical for transcription activation and replication initiator activities of E2. Therefore, we suspected that Brd4 might have a more complex role in the PV life cycle than initially proposed. We tested this idea in the present study and show that the Brd4 bromodomain protein can indeed participate in the BPV1 E2-dependent transcription activation and DNA replication processes. Brd4 is specifically involved in the E2-activated transcription process; the role of Brd4 in BPV1 DNA replication, however, is either largely or completely independent of its binding to E2. Our data demonstrate the possible involvement of Brd4 also in polyomavirus DNA replication and reveal the varying importance of the Brd4-linked component for BPV1 DNA replication in different cell lines.
Cloning of the dominant-negative form of Brd4 (Brd4 CTD). The use of a dominant-negative truncated version of Brd4 is a useful alternative to manipulations with a full-length gene, as overexpression or knockout of Brd4 in mammalian cells has been shown to cause severe alterations in cell growth (5, 15). Overexpression of Brd4 CTD affects neither the growth of several cell lines (C127, C33A, HeLa) (25, 26) nor the cell cycle distribution of CHO cells stably expressing BPV1 E2 (I. Ilves, and M. Ustav, unpublished data). E2 is the only known target of the C-terminal domain of Brd4; all interactions of Brd4 with other proteins identified so far are carried out through its N-terminal bromodomain-containing part. We cloned the CTD (last 315 amino acids) of human Brd4 into a pCG expression vector (21). The N terminus of the resulting protein also carries a BPV1 E2-derived E2Tag epitope tag, which enables the estimation of the E2:CTD ratio in our experiments (9), and a nuclear localization signal from the simian virus 40 (SV40) large T antigen. This Brd4 CTD protein coimmunoprecipitated efficiently with BPV1 E2 from lysates of transfected Cos7 cells when the antibodies that recognized E2 but not the epitope-tagged CTD were used. Immunofluorescence analysis revealed clearly nuclear localization of the overexpressed protein (data not shown).
Brd4 CTD inhibits transient replication of BPV1 genomic DNA. We tested the effect of Brd4 CTD on transient BPV1 genome replication in C127 cells, in a system that essentially mimics the initial step of the BPV1 transformation assay that has long been used as a model for studying the nonproductive part of the BPV1 life cycle (6). We extracted low-molecular-weight DNA on days 2 and 3 after transfection of cells by electroporation, digested it with HindIII to linearize the BPV1 genome, and detected newly replicated BPV1 DNA using DpnI endonuclease digestion (which separates bacterially methylated, unreplicated input DNA) followed by Southern blotting (22) (Fig. 1). Replication of the BPV1 genome was inhibited in cells cotransfected with 1 μg (Fig. 1, lanes 4 and 5) or 3 μg (lanes 6 and 7) of CTD expression vector compared to control transfections with either the same amounts of control vector (lanes 8 to 11) or BPV1 DNA alone (lanes 2 and 3). pCGdXS, used as a control vector here and in the following experiments, was identical to Brd4 CTD vector pCGCTD except that it expressed 100-bp-long nonsense mRNA without any functional open reading frames. The effect of the CTD on BPV1 replication in our experiments was unlikely to be due to interference with the E2/Brd4-dependent chromosome attachment and mitotic partitioning process, as we showed earlier that the replication efficiency of BPV1 origin-containing reporter plasmids in short-term assays is independent of their tethering to host chromosomes (7). As discussed above, Brd4 CTD does not affect the cell growth or division cycle, and the possibility of an indirect effect of Brd4 CTD on BPV1 replication caused by changes in these processes was also unlikely. We decided to test two remaining possibilities: first, that the CTD interfered directly with the BPV1 DNA replication, and second, that it suppressed the E2-activated transcription of essential viral replication proteins E1 and E2.
Brd4 CTD inhibits BPV1 DNA replication and does so independently of its binding to E2. To test for a direct effect of Brd4 CTD on the BPV1 DNA replication, we cotransfected cells with a reporter plasmid containing the BPV1 origin of replication (ori), expression constructs for BPV1 replication proteins E1 and E2, and either CTD-expressing or control plasmid. This way, we could test the effect of Brd4 CTD in a simple model which consists of minimal cis and trans determinants of viral replication. The exact amounts of transfected plasmid DNA here and in the following series with different cell lines were chosen on the basis of preliminary experiments, to ensure that the levels of E2 and Brd4 CTD as well as the CTD:E2 ratio were comparable in all experiments. The detection of newly replicated reporter DNA was performed essentially as described above for BPV1 genome replication experiments. The amount of newly replicated reporter plasmid DNA was clearly lower in C127 and CHO cells cotransfected with CTD expression construct (Fig. 2A, lanes 4, 5, 13, and 14) than in cells cotransfected with the same amount of control vector (lanes 2, 3, 11, and 12). This effect of Brd4 CTD on BPV1 ori replication is not due to the lower expression of the viral replication proteins (see the level of E2 in a parallel Western blot [Fig. 2B, compare +CTD lanes to –CTD lanes). In contrast, we have noticed that E2 levels tend to be even higher when E2 is expressed together with Brd4 CTD (see also Fig. 3C and text below). We were unable to detect the E1 protein in our experiments due to its very low levels. However, CTD was unlikely to suppress E1 expression, as both E1 and E2 were expressed from cytomegalovirus promoters in identical pCG vector constructs. In addition, we could not detect any significant effect of Brd4 CTD on the expression of LTAg or VP16E2 proteins from the same vector (see Fig. 2D and 3C and text below). To our surprise, CTD was unable to inhibit the replication of BPV1 ori reporter in human C33A cells, where the interaction between E2 and Brd4 was first observed (25) (Fig. 2A, compare lanes 22 and 23 to lanes 20 and 21). According to parallel Western blotting analysis with a horseradish peroxidase-conjugated anti-E2Tag antibody that recognizes a single epitope in both E2 and epitope-tagged CTD proteins, the levels of Brd4 CTD and E2 were roughly similar in C33A cells and in C127 and CHO cells, where the CTD acted as an efficient inhibitor of BPV1 DNA replication (Fig. 2B, compare lane 12 to lane 3 or 8, respectively; note the uppermost, nonspecific band on all Western blots, which we have found to serve as good internal reference for rough estimation of the relative signal strength in the cell lines used). Moreover, the CTD certainly not only binds to E2 in C33A cells but also can behave as a dominant-negative inhibitor of other Brd4-related activities of E2 in this cell line: its ectopic expression excludes E2 from chromatin (25) and, as we show below, inhibits E2-dependent transcription activation. This led us to suspect that the inhibition of the BPV1 DNA replication by Brd4 CTD that we observed in C127 and CHO cells could have been achieved independently of the binding of the CTD to E2.
In order to test this possibility, we replaced wild-type (wt) E2 with a mutant version, 37/73 (arginine 37 and isoleucine 73 replaced by alanine), in our transient-replication experiments. This protein supports BPV1 DNA replication efficiently but does not bind to Brd4 (2). 37/73 was expressed at lower levels than wt E2 in CHO cells (Fig. 2B, lanes 9 and 10) and especially in C127 cells (lanes 4 and 5), where it was barely detectable in Western blots. 37/73 also supported lower levels of BPV1 ori replication in CHO cells (Fig. 2A, compare lanes 15 and 16 and lanes 11 and 12; it should be noted that the difference was smaller in some experiments) but, surprisingly, was even more effective than wt E2 in C127 cells (Fig. 2A, compare lanes 6 and 7 and lanes 2 and 3). Despite these variations between cell lines, the replication of BPV1 ori supported by 37/73 was equally sensitive to Brd4 CTD in C127 and CHO cells (Fig. 2A, compare lanes 8 and 9 to lanes 6 and 7 and lanes 17 and 18 to lanes 15 and 16, respectively). Therefore, the dominant-negative effect of Brd4 CTD on the BPV1 ori dependent replication does not require its binding to E2 and either largely or completely targets some other component(s) of the replication initiation complex.
The inhibitory effect of Brd4 CTD on viral DNA replication is not limited to BPV1. The fact that inhibition of BPV1 DNA replication by Brd4 CTD was carried out independently of its binding to the virus-specific replication factor (E2) led us to consider the possibility that BPV1 might not be the only DNA virus with a Brd4-linked replication mechanism. We tested this idea on mouse polyomavirus (Py) DNA replication, using a transient-replication assay similar to the one that we used to detect the involvement of Brd4 in BPV1 DNA replication (see above). We transfected C127 cells with the expression vector for Py replication initiator large T antigen (LTAg), a reporter plasmid carrying either wild-type Py (Fig. 2C, lanes 1 to 4) or core (enhancerless) ori (lanes 5 to 8), and either a CTD-expressing vector (lanes 3, 4, 7, and 8) or a control vector (lanes 1, 2, 5, and 6). The hybridization signal corresponding to the newly replicated Py ori reporter DNA was significantly lower in the cells expressing CTD, independent of the Py ori configuration (compare lanes 3 and 4 and lanes 7 and 8 to lanes 1 and 2 and lanes 5 and 6, respectively). Parallel Western blots did not show any significant loss of LT expression, which could have explained such an effect on Py ori replication (Fig. 2D). We can conclude from these data that the dominant-negative effect of Brd4 CTD on the viral DNA replication is not limited to papillomaviruses and most likely targets some general component of the replication machinery.
Brd4 CTD inhibits E2-dependent transcription activation. E2 stimulates transcription from early promoters of BPV1 through sequences located in the upstream regulatory region (URR) of the viral genome (20). To test the possibility that Brd4 CTD could have interfered with the BPV1 genomic replication by inhibiting the E2-activated transcription of viral replication factors, we made use of the dual-luciferase reporter assay system from Promega (Madison, WI). We transfected cells with expression constructs for E2 and Brd4 CTD as well as with two different luciferase reporter plasmids, one expressing firefly luciferase under the control of URR fragment (nucleotides 7476 to 7494 from the BPV1 genome) and one expressing Renilla reniformis luciferase from the herpes simplex virus thymidine kinase promoter. The cells were harvested for detection of both luciferase activities, as a measure of respective expression levels, on day 2 after transfection, according to a standard protocol provided by manufacturer. The data for E2-responsive firefly luciferase expression were normalized to the data for non-E2-responsive Renilla luciferase expression, and the results were presented relative to the respective value from the samples corresponding to E2-activated transcription (cotransfection of the E2 expression plasmid with pCGdXS control vector [Fig. 3A, +E2 –CTD columns). Our results show that E2-activated transcription is efficiently down-regulated by Brd4 CTD. The cotransfection of Brd4 CTD-expressing vector inhibited the E2-dependent transactivation virtually down to basal levels in C127 cells (Fig. 3A, compare +E2 –CTD, +E2 +CTD, and basal columns). The inhibition appears also in CHO and C33A cells, even though it is somewhat weaker in the latter case (Fig. 3A). A similar CTD-dependent inhibitory effect on the E2-dependent transactivation was apparent in experiments where a reporter with an artificial E2-dependent promoter construct (enhancerless SV40 early promoter linked to three E2 binding sites) was used instead of the BPV1 native promoter (data not shown). The comparison of the data from the same series normalized either to the E2-independent Renilla luciferase expression or to the total protein concentration in the samples revealed the same overall pattern within the series (data not shown). The expression of Brd4 CTD did not have any detectable effect on the basal (without E2 activation) transcription of luciferase from the BPV1 native promoter construct in C127 and CHO cells (Fig. 3A, compare the +CTD and basal columns). Similarly, no effect of CTD was detected on firefly luciferase expression from the mammalian HSP70 promoter, or when the Renilla luciferase expression data from the non-E2-responsive thymidine kinase promoter were normalized to the total protein concentration (data not shown). We conclude from these results that the overexpression of Brd4 CTD does not have any unspecific gross effects on general cellular transcription and specifically targets the transcription activated by E2.
To further show that the effect of Brd4 CTD was indeed specific to E2-activated transcription, we performed experiments where increasing amounts of the Brd4 CTD expression construct were cotransfected with luciferase reporters as well as with pCG vector expressing either BPV1 wt E2 or VP16E2 fusion protein (Fig. 3B). VP16E2 contains the BPV1 E2 DNA binding domain fused to the strong transactivation domain from the VP16 protein of human herpesvirus 1 (14) and thus effectively activates transcription from promoters that contain E2 binding sites. We found that E2-activated transcription from BPV1 native promoter reporter was effectively inhibited by CTD expression in a concentration-dependent fashion in both C127 and CHO cells (Fig. 3B, shaded columns; columns labeled "0" on the same graphs represent control transfections with no CTD), while transcription activated from the same reporter by VP16E2 was not comparably affected by cotransfection of the same concentrations of Brd4 CTD vector (open columns on the same graphs). Under conditions in which the E2 and CTD expression levels and ratio were roughly similar in C127 and CHO cells (Fig. 3C, compare wtE2 lanes in the upper blots), the effect of Brd4 CTD was especially strong in C127 cells. In these cells, the addition of 30 ng CTD expression plasmid, which results in approximately equal molar quantities of CTD and E2 (Fig. 3C, lane 4 in the bottom Western blot), leads to the loss of E2-dependent activation by more than 60% (Fig. 3B, compare dark columns 30 and 0). The addition of 3,000 ng of CTD expression construct resulted in an almost complete loss of E2-dependent activation in C127 cells (compare columns 3000 and 0). We can conclude from these data that the negative effect of CTD on E2-activated transcription targets the N-terminal transactivation (and Brd4 binding) domain of E2, but not its C-terminal DNA binding domain, E2 binding sites in the promoter region, or activated transcription in general. As in the case of control Western blot analysis for the replication experiments described above, we noticed that the expression of CTD was accompanied by a higher expression level of E2, in a concentration-dependent fashion (Fig. 3C, lanes wtE2). On the other hand, the level of VP16E2 protein remained constant despite Brd4 CTD coexpression (Fig. 3C, VP16E2 lanes). The only effect of Brd4 CTD on transcription that we were able to detect involves the E2-activated promoters. Therefore, even though we have not studied this phenomenon in more detail, the observed rise in the level of wt E2 is most likely due to posttranscriptional effect and could reflect the stabilization of E2 protein due to its binding to Brd4 CTD. In this context, it is interesting that the interaction of BPV1 E2 protein with Brd4 has been reported to stabilize its association with chromatin, even though it was not specified if it was due to the E2 protein stabilization, the tighter interaction of E2 with chromatin, or both (16). For reasons that are unclear at the moment, the positive effect of CTD on the level of E2 seemed to be somewhat weaker in cells expressing high CTD concentrations (Fig. 3C, lanes 6 and 17).
In summary, we show here that the overexpression of Brd4 CTD has a dominant-negative effect on the replication of BPV1 genome in vivo and that this effect is likely to be carried out through several E2-dependent and -independent pathways simultaneously. These results suggest a complex role for Brd4 in the BPV1 life cycle and expose the E2-Brd4 interaction as an important target for the development of antiviral drugs. Several interesting perspectives can also be proposed concerning the mechanisms of PV replication and E2-dependent transcriptional activation.
The dominant-negative phenotype that is caused by Brd4 CTD may correspond to two opposite possibilities: first, that the binding of full-length Brd4 to certain targets through its C-terminal domain is required for the normal phenotype and the truncated CTD version interferes with such binding, or second, that overexpression of the CTD mimics the negative effect of binding by full-length Brd4 that might also occur in normal cells. We currently do not have enough data to speculate which of these possibilities explains the inhibition of transient replication of the PV origin by Brd4 CTD. Brd4 has been shown to interact with cellular replication factor C (RFC), but this binding requires the N-terminal bromodomain-containing region of Brd4, which is missing from the CTD (15). We do know, however, that the role of Brd4 is not limited to DNA replication of papillomaviruses and can be carried out independently of its binding to E2, most likely involving some general component of the replication process. Our data thus indicate that Brd4 might be involved in steps of cellular DNA replication other than those linked to RFC binding. Considering the apparent insensitivity of the cell division cycle to Brd4 CTD, this role of Brd4 is not critical for the cellular DNA replication. Interestingly, this Brd4-linked step also seems to be of varied importance for BPV1 DNA replication, as shown by the lack of Brd4 CTD-dependent inhibition of BPV1 DNA replication in C33A cells. Such variability in certain details of the BPV1 replication mechanism is supported by an earlier study that demonstrated its cell-specific inhibition by p53 (13).
Previous genetic analysis of BPV1 E2 activities has demonstrated relatively good general correlation between its transactivation properties and binding to Brd4, suggesting that overlapping structural determinants could be required for both functions of E2 (2). This correlation was not perfect, however. Our present study used the dominant-negative effect of the E2-interacting part of Brd4 to show unequivocally that direct interaction with Brd4 can modulate the transactivation properties of E2 protein. Brd4 has recently been shown to interact with components of the Mediator coactivator complex and can recruit the p-TEFb elongation regulating complex to promoter regions (8, 24). These data and those presented here strongly suggest the involvement of Brd4 and associated complexes, like p-TEFb and Mediator, in transcription activation by BPV1 E2. The detailed mechanism for such activation remains to be shown. For example, E2 may directly recruit Brd4 and associated activating complexes to promoters adjacent to E2 binding sites, or it might facilitate the recruitment of Brd4 to acetylated histones in the promoter region. Our data suggest that the Brd4-mediated mechanism is not generally used in the transcriptional activation process, as the transcription activated by VP16 transactivation domain was insensitive to CTD. It remains to be seen if there are any other transcription regulators besides E2 that use such a Brd4-dependent mechanism, and also if there are any alternative mechanisms for E2-dependent transactivation and how these are coordinated with each other. The interaction of E2 with Brd4 persists throughout the cell cycle (11, 16), and the transactivation and chromatin attachment activities of E2 are thus likely to be executed by heavily overlapping Brd4-containing complexes. It means that these two activities of E2 have to be tightly coordinated with each other. This conclusion is backed by the earlier genetic analyses of E2, which demonstrated relatively good general correlation between its transactivation and chromatin attachment activities (1, 2). However, few specific mutations in the N-terminal domain were found in these studies that could still make E2 behave somewhat differently in transactivation and chromosome binding functions, indicating that certain differences do exist in respective molecular complexes.
Finally, in light of our data, the previously reported dominant-negative effect of Brd4 CTD on the transformation of C127 cells (25) is unlikely to be caused solely by affecting E2-Brd4-dependent chromatin attachment and partitioning of viral genomes. Several processes are affected simultaneously in such assays, and it is extremely difficult to pinpoint the exact role for each individual process in the overall outcome. Our attempts to demonstrate the inhibitory effect of Brd4 CTD on E2-dependent extrachromosomal plasmid maintenance in simple but effective green fluorescent protein-based replication-free partitioning assays (1) have been unsuccessful (I. Ilves, K. Memets, and M. Ustav, unpublished data). One possible explanation is that this was simply due to insufficient sensitivity of the assay system. Another possibility, however, is that additional chromatin receptors or components of the receptor complex for E2 apart from Brd4 could exist. This could also explain why fluorescent in situ hybridization (FISH) analysis reveals almost immediate Brd4 CTD-dependent dissociation of the viral DNA from chromosomes, but the loss of viral DNA from cells is detectable only after four or more passages of transfected C127 cells at a 1:10 ratio (25, 26). On the other hand, only two to three cell doublings were enough to reveal the inefficiency of the control plasmid maintenance compared to the E2-dependent system in the above-mentioned green fluorescent protein-based partitioning assay (1). It is possible that the loss of interaction with Brd4 could weaken the E2-mediated tethering to chromosomes sufficiently to enable its detection in FISH analysis of fixed cells, but interaction(s) with an additional target(s) could prove to be sufficient to prevent rapid changes in the viral genome copy number in vivo. Future studies should examine more closely the specific complexes and detailed mechanisms involved in the Brd4-dependent processes of the BPV1 life cycle. Such studies may also be a useful tool for elucidating further the specific complexes of Brd4 and its functions in the cell.
ACKNOWLEDGMENTS
We thank Aare Abroi and Reet Kurg for discussions and critical reading of the manuscript and Anne Kalling for technical assistance.
This work was supported by grants SF0182566, ETF5999, and ETF5998 from the Estonian Science Foundation and INTNL55000339 from HHMI.
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Department of Microbiology and Virology, Institute of Molecular and Cell Biology, University of Tartu and Estonian Biocentre, Tartu, Estonia
ABSTRACT
Brd4 protein has been proposed to act as a cellular receptor for the bovine papillomavirus type 1 (BPV1) E2 protein in the E2-mediated chromosome attachment and mitotic segregation of viral genomes. Here, we provide data that show the involvement of Brd4 in multiple early functions of the BPV1 life cycle, suggest a Brd4-dependent mechanism for E2-dependent transcription activation, and indicate the role of Brd4 in papillomavirus and polyomavirus replication as well as cell-specific utilization of Brd4-linked features in BPV1 DNA replication. Our data also show the potential therapeutic value of the disruption of the E2-Brd4 interaction for the development of antiviral drugs.
TEXT
Papillomavirus (PV) E2 protein is a central regulator of the viral life cycle. In addition to its well-established activity as a transcription modulator and replication initiator protein (6), E2 of bovine papillomavirus type 1 (BPV1) has recently emerged as a trans factor which mediates mitotic segregation of viral genomes by tethering them to host cell chromatin (7, 12, 19). The first candidate for a receptor of E2 in the latter process, Brd4, is attached to the chromatin through its two bromodomains, which bind to acetylated histones H3 and H4 both in interphase and in mitosis (4, 25). Mutated E2 proteins that are defective in Brd4 binding are unable to bind to mitotic chromosomes (2), and ectopic expression of Brd4 can reconstitute the BPV1 E2-dependent extrachromosomal plasmid maintenance in the yeast Saccharomyces cerevisiae, where such a process normally does not function (3). Ectopic expression of the E2-binding C-terminal domain (CTD) of Brd4 in mammalian cells disrupts the interaction of E2 with cellular Brd4 and relocates E2 from mitotic chromosomes (25, 26). Brd4 CTD binds to the N-terminal domain of E2 (25), which is also responsible for interactions critical for transcription activation and replication initiator activities of E2. Therefore, we suspected that Brd4 might have a more complex role in the PV life cycle than initially proposed. We tested this idea in the present study and show that the Brd4 bromodomain protein can indeed participate in the BPV1 E2-dependent transcription activation and DNA replication processes. Brd4 is specifically involved in the E2-activated transcription process; the role of Brd4 in BPV1 DNA replication, however, is either largely or completely independent of its binding to E2. Our data demonstrate the possible involvement of Brd4 also in polyomavirus DNA replication and reveal the varying importance of the Brd4-linked component for BPV1 DNA replication in different cell lines.
Cloning of the dominant-negative form of Brd4 (Brd4 CTD). The use of a dominant-negative truncated version of Brd4 is a useful alternative to manipulations with a full-length gene, as overexpression or knockout of Brd4 in mammalian cells has been shown to cause severe alterations in cell growth (5, 15). Overexpression of Brd4 CTD affects neither the growth of several cell lines (C127, C33A, HeLa) (25, 26) nor the cell cycle distribution of CHO cells stably expressing BPV1 E2 (I. Ilves, and M. Ustav, unpublished data). E2 is the only known target of the C-terminal domain of Brd4; all interactions of Brd4 with other proteins identified so far are carried out through its N-terminal bromodomain-containing part. We cloned the CTD (last 315 amino acids) of human Brd4 into a pCG expression vector (21). The N terminus of the resulting protein also carries a BPV1 E2-derived E2Tag epitope tag, which enables the estimation of the E2:CTD ratio in our experiments (9), and a nuclear localization signal from the simian virus 40 (SV40) large T antigen. This Brd4 CTD protein coimmunoprecipitated efficiently with BPV1 E2 from lysates of transfected Cos7 cells when the antibodies that recognized E2 but not the epitope-tagged CTD were used. Immunofluorescence analysis revealed clearly nuclear localization of the overexpressed protein (data not shown).
Brd4 CTD inhibits transient replication of BPV1 genomic DNA. We tested the effect of Brd4 CTD on transient BPV1 genome replication in C127 cells, in a system that essentially mimics the initial step of the BPV1 transformation assay that has long been used as a model for studying the nonproductive part of the BPV1 life cycle (6). We extracted low-molecular-weight DNA on days 2 and 3 after transfection of cells by electroporation, digested it with HindIII to linearize the BPV1 genome, and detected newly replicated BPV1 DNA using DpnI endonuclease digestion (which separates bacterially methylated, unreplicated input DNA) followed by Southern blotting (22) (Fig. 1). Replication of the BPV1 genome was inhibited in cells cotransfected with 1 μg (Fig. 1, lanes 4 and 5) or 3 μg (lanes 6 and 7) of CTD expression vector compared to control transfections with either the same amounts of control vector (lanes 8 to 11) or BPV1 DNA alone (lanes 2 and 3). pCGdXS, used as a control vector here and in the following experiments, was identical to Brd4 CTD vector pCGCTD except that it expressed 100-bp-long nonsense mRNA without any functional open reading frames. The effect of the CTD on BPV1 replication in our experiments was unlikely to be due to interference with the E2/Brd4-dependent chromosome attachment and mitotic partitioning process, as we showed earlier that the replication efficiency of BPV1 origin-containing reporter plasmids in short-term assays is independent of their tethering to host chromosomes (7). As discussed above, Brd4 CTD does not affect the cell growth or division cycle, and the possibility of an indirect effect of Brd4 CTD on BPV1 replication caused by changes in these processes was also unlikely. We decided to test two remaining possibilities: first, that the CTD interfered directly with the BPV1 DNA replication, and second, that it suppressed the E2-activated transcription of essential viral replication proteins E1 and E2.
Brd4 CTD inhibits BPV1 DNA replication and does so independently of its binding to E2. To test for a direct effect of Brd4 CTD on the BPV1 DNA replication, we cotransfected cells with a reporter plasmid containing the BPV1 origin of replication (ori), expression constructs for BPV1 replication proteins E1 and E2, and either CTD-expressing or control plasmid. This way, we could test the effect of Brd4 CTD in a simple model which consists of minimal cis and trans determinants of viral replication. The exact amounts of transfected plasmid DNA here and in the following series with different cell lines were chosen on the basis of preliminary experiments, to ensure that the levels of E2 and Brd4 CTD as well as the CTD:E2 ratio were comparable in all experiments. The detection of newly replicated reporter DNA was performed essentially as described above for BPV1 genome replication experiments. The amount of newly replicated reporter plasmid DNA was clearly lower in C127 and CHO cells cotransfected with CTD expression construct (Fig. 2A, lanes 4, 5, 13, and 14) than in cells cotransfected with the same amount of control vector (lanes 2, 3, 11, and 12). This effect of Brd4 CTD on BPV1 ori replication is not due to the lower expression of the viral replication proteins (see the level of E2 in a parallel Western blot [Fig. 2B, compare +CTD lanes to –CTD lanes). In contrast, we have noticed that E2 levels tend to be even higher when E2 is expressed together with Brd4 CTD (see also Fig. 3C and text below). We were unable to detect the E1 protein in our experiments due to its very low levels. However, CTD was unlikely to suppress E1 expression, as both E1 and E2 were expressed from cytomegalovirus promoters in identical pCG vector constructs. In addition, we could not detect any significant effect of Brd4 CTD on the expression of LTAg or VP16E2 proteins from the same vector (see Fig. 2D and 3C and text below). To our surprise, CTD was unable to inhibit the replication of BPV1 ori reporter in human C33A cells, where the interaction between E2 and Brd4 was first observed (25) (Fig. 2A, compare lanes 22 and 23 to lanes 20 and 21). According to parallel Western blotting analysis with a horseradish peroxidase-conjugated anti-E2Tag antibody that recognizes a single epitope in both E2 and epitope-tagged CTD proteins, the levels of Brd4 CTD and E2 were roughly similar in C33A cells and in C127 and CHO cells, where the CTD acted as an efficient inhibitor of BPV1 DNA replication (Fig. 2B, compare lane 12 to lane 3 or 8, respectively; note the uppermost, nonspecific band on all Western blots, which we have found to serve as good internal reference for rough estimation of the relative signal strength in the cell lines used). Moreover, the CTD certainly not only binds to E2 in C33A cells but also can behave as a dominant-negative inhibitor of other Brd4-related activities of E2 in this cell line: its ectopic expression excludes E2 from chromatin (25) and, as we show below, inhibits E2-dependent transcription activation. This led us to suspect that the inhibition of the BPV1 DNA replication by Brd4 CTD that we observed in C127 and CHO cells could have been achieved independently of the binding of the CTD to E2.
In order to test this possibility, we replaced wild-type (wt) E2 with a mutant version, 37/73 (arginine 37 and isoleucine 73 replaced by alanine), in our transient-replication experiments. This protein supports BPV1 DNA replication efficiently but does not bind to Brd4 (2). 37/73 was expressed at lower levels than wt E2 in CHO cells (Fig. 2B, lanes 9 and 10) and especially in C127 cells (lanes 4 and 5), where it was barely detectable in Western blots. 37/73 also supported lower levels of BPV1 ori replication in CHO cells (Fig. 2A, compare lanes 15 and 16 and lanes 11 and 12; it should be noted that the difference was smaller in some experiments) but, surprisingly, was even more effective than wt E2 in C127 cells (Fig. 2A, compare lanes 6 and 7 and lanes 2 and 3). Despite these variations between cell lines, the replication of BPV1 ori supported by 37/73 was equally sensitive to Brd4 CTD in C127 and CHO cells (Fig. 2A, compare lanes 8 and 9 to lanes 6 and 7 and lanes 17 and 18 to lanes 15 and 16, respectively). Therefore, the dominant-negative effect of Brd4 CTD on the BPV1 ori dependent replication does not require its binding to E2 and either largely or completely targets some other component(s) of the replication initiation complex.
The inhibitory effect of Brd4 CTD on viral DNA replication is not limited to BPV1. The fact that inhibition of BPV1 DNA replication by Brd4 CTD was carried out independently of its binding to the virus-specific replication factor (E2) led us to consider the possibility that BPV1 might not be the only DNA virus with a Brd4-linked replication mechanism. We tested this idea on mouse polyomavirus (Py) DNA replication, using a transient-replication assay similar to the one that we used to detect the involvement of Brd4 in BPV1 DNA replication (see above). We transfected C127 cells with the expression vector for Py replication initiator large T antigen (LTAg), a reporter plasmid carrying either wild-type Py (Fig. 2C, lanes 1 to 4) or core (enhancerless) ori (lanes 5 to 8), and either a CTD-expressing vector (lanes 3, 4, 7, and 8) or a control vector (lanes 1, 2, 5, and 6). The hybridization signal corresponding to the newly replicated Py ori reporter DNA was significantly lower in the cells expressing CTD, independent of the Py ori configuration (compare lanes 3 and 4 and lanes 7 and 8 to lanes 1 and 2 and lanes 5 and 6, respectively). Parallel Western blots did not show any significant loss of LT expression, which could have explained such an effect on Py ori replication (Fig. 2D). We can conclude from these data that the dominant-negative effect of Brd4 CTD on the viral DNA replication is not limited to papillomaviruses and most likely targets some general component of the replication machinery.
Brd4 CTD inhibits E2-dependent transcription activation. E2 stimulates transcription from early promoters of BPV1 through sequences located in the upstream regulatory region (URR) of the viral genome (20). To test the possibility that Brd4 CTD could have interfered with the BPV1 genomic replication by inhibiting the E2-activated transcription of viral replication factors, we made use of the dual-luciferase reporter assay system from Promega (Madison, WI). We transfected cells with expression constructs for E2 and Brd4 CTD as well as with two different luciferase reporter plasmids, one expressing firefly luciferase under the control of URR fragment (nucleotides 7476 to 7494 from the BPV1 genome) and one expressing Renilla reniformis luciferase from the herpes simplex virus thymidine kinase promoter. The cells were harvested for detection of both luciferase activities, as a measure of respective expression levels, on day 2 after transfection, according to a standard protocol provided by manufacturer. The data for E2-responsive firefly luciferase expression were normalized to the data for non-E2-responsive Renilla luciferase expression, and the results were presented relative to the respective value from the samples corresponding to E2-activated transcription (cotransfection of the E2 expression plasmid with pCGdXS control vector [Fig. 3A, +E2 –CTD columns). Our results show that E2-activated transcription is efficiently down-regulated by Brd4 CTD. The cotransfection of Brd4 CTD-expressing vector inhibited the E2-dependent transactivation virtually down to basal levels in C127 cells (Fig. 3A, compare +E2 –CTD, +E2 +CTD, and basal columns). The inhibition appears also in CHO and C33A cells, even though it is somewhat weaker in the latter case (Fig. 3A). A similar CTD-dependent inhibitory effect on the E2-dependent transactivation was apparent in experiments where a reporter with an artificial E2-dependent promoter construct (enhancerless SV40 early promoter linked to three E2 binding sites) was used instead of the BPV1 native promoter (data not shown). The comparison of the data from the same series normalized either to the E2-independent Renilla luciferase expression or to the total protein concentration in the samples revealed the same overall pattern within the series (data not shown). The expression of Brd4 CTD did not have any detectable effect on the basal (without E2 activation) transcription of luciferase from the BPV1 native promoter construct in C127 and CHO cells (Fig. 3A, compare the +CTD and basal columns). Similarly, no effect of CTD was detected on firefly luciferase expression from the mammalian HSP70 promoter, or when the Renilla luciferase expression data from the non-E2-responsive thymidine kinase promoter were normalized to the total protein concentration (data not shown). We conclude from these results that the overexpression of Brd4 CTD does not have any unspecific gross effects on general cellular transcription and specifically targets the transcription activated by E2.
To further show that the effect of Brd4 CTD was indeed specific to E2-activated transcription, we performed experiments where increasing amounts of the Brd4 CTD expression construct were cotransfected with luciferase reporters as well as with pCG vector expressing either BPV1 wt E2 or VP16E2 fusion protein (Fig. 3B). VP16E2 contains the BPV1 E2 DNA binding domain fused to the strong transactivation domain from the VP16 protein of human herpesvirus 1 (14) and thus effectively activates transcription from promoters that contain E2 binding sites. We found that E2-activated transcription from BPV1 native promoter reporter was effectively inhibited by CTD expression in a concentration-dependent fashion in both C127 and CHO cells (Fig. 3B, shaded columns; columns labeled "0" on the same graphs represent control transfections with no CTD), while transcription activated from the same reporter by VP16E2 was not comparably affected by cotransfection of the same concentrations of Brd4 CTD vector (open columns on the same graphs). Under conditions in which the E2 and CTD expression levels and ratio were roughly similar in C127 and CHO cells (Fig. 3C, compare wtE2 lanes in the upper blots), the effect of Brd4 CTD was especially strong in C127 cells. In these cells, the addition of 30 ng CTD expression plasmid, which results in approximately equal molar quantities of CTD and E2 (Fig. 3C, lane 4 in the bottom Western blot), leads to the loss of E2-dependent activation by more than 60% (Fig. 3B, compare dark columns 30 and 0). The addition of 3,000 ng of CTD expression construct resulted in an almost complete loss of E2-dependent activation in C127 cells (compare columns 3000 and 0). We can conclude from these data that the negative effect of CTD on E2-activated transcription targets the N-terminal transactivation (and Brd4 binding) domain of E2, but not its C-terminal DNA binding domain, E2 binding sites in the promoter region, or activated transcription in general. As in the case of control Western blot analysis for the replication experiments described above, we noticed that the expression of CTD was accompanied by a higher expression level of E2, in a concentration-dependent fashion (Fig. 3C, lanes wtE2). On the other hand, the level of VP16E2 protein remained constant despite Brd4 CTD coexpression (Fig. 3C, VP16E2 lanes). The only effect of Brd4 CTD on transcription that we were able to detect involves the E2-activated promoters. Therefore, even though we have not studied this phenomenon in more detail, the observed rise in the level of wt E2 is most likely due to posttranscriptional effect and could reflect the stabilization of E2 protein due to its binding to Brd4 CTD. In this context, it is interesting that the interaction of BPV1 E2 protein with Brd4 has been reported to stabilize its association with chromatin, even though it was not specified if it was due to the E2 protein stabilization, the tighter interaction of E2 with chromatin, or both (16). For reasons that are unclear at the moment, the positive effect of CTD on the level of E2 seemed to be somewhat weaker in cells expressing high CTD concentrations (Fig. 3C, lanes 6 and 17).
In summary, we show here that the overexpression of Brd4 CTD has a dominant-negative effect on the replication of BPV1 genome in vivo and that this effect is likely to be carried out through several E2-dependent and -independent pathways simultaneously. These results suggest a complex role for Brd4 in the BPV1 life cycle and expose the E2-Brd4 interaction as an important target for the development of antiviral drugs. Several interesting perspectives can also be proposed concerning the mechanisms of PV replication and E2-dependent transcriptional activation.
The dominant-negative phenotype that is caused by Brd4 CTD may correspond to two opposite possibilities: first, that the binding of full-length Brd4 to certain targets through its C-terminal domain is required for the normal phenotype and the truncated CTD version interferes with such binding, or second, that overexpression of the CTD mimics the negative effect of binding by full-length Brd4 that might also occur in normal cells. We currently do not have enough data to speculate which of these possibilities explains the inhibition of transient replication of the PV origin by Brd4 CTD. Brd4 has been shown to interact with cellular replication factor C (RFC), but this binding requires the N-terminal bromodomain-containing region of Brd4, which is missing from the CTD (15). We do know, however, that the role of Brd4 is not limited to DNA replication of papillomaviruses and can be carried out independently of its binding to E2, most likely involving some general component of the replication process. Our data thus indicate that Brd4 might be involved in steps of cellular DNA replication other than those linked to RFC binding. Considering the apparent insensitivity of the cell division cycle to Brd4 CTD, this role of Brd4 is not critical for the cellular DNA replication. Interestingly, this Brd4-linked step also seems to be of varied importance for BPV1 DNA replication, as shown by the lack of Brd4 CTD-dependent inhibition of BPV1 DNA replication in C33A cells. Such variability in certain details of the BPV1 replication mechanism is supported by an earlier study that demonstrated its cell-specific inhibition by p53 (13).
Previous genetic analysis of BPV1 E2 activities has demonstrated relatively good general correlation between its transactivation properties and binding to Brd4, suggesting that overlapping structural determinants could be required for both functions of E2 (2). This correlation was not perfect, however. Our present study used the dominant-negative effect of the E2-interacting part of Brd4 to show unequivocally that direct interaction with Brd4 can modulate the transactivation properties of E2 protein. Brd4 has recently been shown to interact with components of the Mediator coactivator complex and can recruit the p-TEFb elongation regulating complex to promoter regions (8, 24). These data and those presented here strongly suggest the involvement of Brd4 and associated complexes, like p-TEFb and Mediator, in transcription activation by BPV1 E2. The detailed mechanism for such activation remains to be shown. For example, E2 may directly recruit Brd4 and associated activating complexes to promoters adjacent to E2 binding sites, or it might facilitate the recruitment of Brd4 to acetylated histones in the promoter region. Our data suggest that the Brd4-mediated mechanism is not generally used in the transcriptional activation process, as the transcription activated by VP16 transactivation domain was insensitive to CTD. It remains to be seen if there are any other transcription regulators besides E2 that use such a Brd4-dependent mechanism, and also if there are any alternative mechanisms for E2-dependent transactivation and how these are coordinated with each other. The interaction of E2 with Brd4 persists throughout the cell cycle (11, 16), and the transactivation and chromatin attachment activities of E2 are thus likely to be executed by heavily overlapping Brd4-containing complexes. It means that these two activities of E2 have to be tightly coordinated with each other. This conclusion is backed by the earlier genetic analyses of E2, which demonstrated relatively good general correlation between its transactivation and chromatin attachment activities (1, 2). However, few specific mutations in the N-terminal domain were found in these studies that could still make E2 behave somewhat differently in transactivation and chromosome binding functions, indicating that certain differences do exist in respective molecular complexes.
Finally, in light of our data, the previously reported dominant-negative effect of Brd4 CTD on the transformation of C127 cells (25) is unlikely to be caused solely by affecting E2-Brd4-dependent chromatin attachment and partitioning of viral genomes. Several processes are affected simultaneously in such assays, and it is extremely difficult to pinpoint the exact role for each individual process in the overall outcome. Our attempts to demonstrate the inhibitory effect of Brd4 CTD on E2-dependent extrachromosomal plasmid maintenance in simple but effective green fluorescent protein-based replication-free partitioning assays (1) have been unsuccessful (I. Ilves, K. Memets, and M. Ustav, unpublished data). One possible explanation is that this was simply due to insufficient sensitivity of the assay system. Another possibility, however, is that additional chromatin receptors or components of the receptor complex for E2 apart from Brd4 could exist. This could also explain why fluorescent in situ hybridization (FISH) analysis reveals almost immediate Brd4 CTD-dependent dissociation of the viral DNA from chromosomes, but the loss of viral DNA from cells is detectable only after four or more passages of transfected C127 cells at a 1:10 ratio (25, 26). On the other hand, only two to three cell doublings were enough to reveal the inefficiency of the control plasmid maintenance compared to the E2-dependent system in the above-mentioned green fluorescent protein-based partitioning assay (1). It is possible that the loss of interaction with Brd4 could weaken the E2-mediated tethering to chromosomes sufficiently to enable its detection in FISH analysis of fixed cells, but interaction(s) with an additional target(s) could prove to be sufficient to prevent rapid changes in the viral genome copy number in vivo. Future studies should examine more closely the specific complexes and detailed mechanisms involved in the Brd4-dependent processes of the BPV1 life cycle. Such studies may also be a useful tool for elucidating further the specific complexes of Brd4 and its functions in the cell.
ACKNOWLEDGMENTS
We thank Aare Abroi and Reet Kurg for discussions and critical reading of the manuscript and Anne Kalling for technical assistance.
This work was supported by grants SF0182566, ETF5999, and ETF5998 from the Estonian Science Foundation and INTNL55000339 from HHMI.
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