Identification and Characterization of CCAAT Enhancer-Binding Protein (C/EBP) as a Transcriptional Activator for Epstein-Barr virus Oncogene Latent Membrane Protein 1

Results: C/EBP was newly isolated to enhance LMP1 promoter in our transient assay system. Conclusion: C/EBP transactivate LMP1 promoter at physiological levels.


LMP1 transcription from both LMP1 promoters in epithelial cells. In conclusion, C/EBP is a newly
Two promoters regulate LMP1 gene transcription, with mechanisms which differ between type II and type III infection. In latency III lymphocyte infection, LMP1 transcription is turned on by EBNA2 and EBNALP from the ED-L1 promoter (5)(6)(7). Although EBNA2 does not feature DNA binding activity, it enhances LMP1 promoter activity by acting as a cofactor. It associates with cellular transcriptional factors, including RBP-Jκ and PU.1, which are then recruited onto the LMP1 promoter for transactivation. EBNA-LP also associates with the complex and further helps the activation process (8).
On the other hand, LMP1 is expressed in an EBNA2-independent manner in type II latency, since neither EBNA2 nor EBNA-LP are available in such type II cells. It has been frequently reported that cytokines, such as IL-4, IL-6, IL-10, IL-13 and IL-21, activate the JAK/STAT pathway, thereby inducing LMP1 gene expression through STAT (9)(10)(11)(12)(13)(14). In certain latency II infected cells including NPC cells, LMP1 transcription originates from a STAT regulated upstream promoter, termed TR-L1, located within the terminal repeats (TR), in addition to the proximal ED-L1 promoter (10,13,15,16). Involvement of transcriptional factors, such as ATF/CREB (17), Sp1/3 (18) and IRF7 (19), has also been indicated. Despite the presence of these well-targeted, focused reports, there is still a possibility of other yet-unknown factor(s) that play(s) essential roles in EBNA2-independent LMP1 expression, because exhaustive investigations have hitherto not been performed.
In the present study, we therefore adopted a comprehensive approach and screened a cDNA library for cellular factors that can activate LMP1 transcription.
We newly cloned the CCAAT Enhancer-Binding Protein (C/EBP) family transcription factor that augments both proximal and distal promoter activation of LMP1 by binding to a motif in the proximal promoter. A functional C/EBP binding site for the LMP1 promoter was identified by reporter mutagenesis and EMSA assays. We also constructed a mutant EBV with a point mutation in the C/EBP binding site, and confirmed importance of binding for LMP1 expression in latent cells.
C666-1 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum. To prepare Hela-CR2/GFP-EBV cells, an EBV-negative Hela cells were stably transformed with CR2 (CD21, the receptor for the EBV expression vector, and infected with GFP-EBV (20). AGS-CR2 was prepared by retroviral transduction of the viral receptor CR2 (CD21) into AGS cells. Akata(-) cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum. Anti-Flag, -C/EBPα and -tubulin antibodies were purchased from Sigma, Cell Signaling and Santa Cruz, respectively. The anti-LMP1 antibody has been described previously (21). Horseradish peroxidase-linked goat antibodies to mouse /rabbit IgG were from Amersham Biosciences.
Library and Plasmids-A SuperScript Pre-made cDNA library (from Human Bone Marrow) was purchased from Invitrogen and used for screening after exclusion of clones with junk inserts. Control reporter pCMV-Rluc was reported previously (22). For pLMP1/ED-L1-Fluc, the ED-L1 promoter sequence of LMP1 was amplified from B95-8 genome using ED-L1pFor and ED-L1pRev primers (Table S1). The amplified DNA was digested with XhoI and NcoI, and then inserted into the XhoI/NcoI sites of pGL4.10 (Promega). Likewise, luciferase reporter constructs containing various TR sequences were prepared using the following primers: for pLMP1/ED-L1+TR-L1-Fluc, ED- L1p+TR-L1pFor and ED-L1p+TR-L1pRev, for   pLMP1/TR-L1-Fluc, TR-L1pFor and TR-L1pRev,   for pLMP1/TR-L1+BS-Fluc, TR-L1p+BSFor and   TR-L1p+BSRev (Table S1). Truncated or point-mutated derivatives of the reporter, pLMP1/ED-L1-Fluc, were made by the inverse PCR method using primers shown in Table S1 (From 417For to 268mtRev). The C/EBP expression vectors were made by inserting cDNA fragments of the proteins into EcoRI/XhoI sites of pcDNA3-Flag (23). RNA was obtained from Akata or HEK293T cells, and subjected to RT-PCR using the SuperScript III First-strand System (Invitrogen) and KOD DNA polymerase (TOYOBO). Primers used for the PCR were listed in the Table S1.  Table S2.

Transfection, Luciferase assay and Immunoblotting -
Electromobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) -EMSA was carried out as described previously (25). Flag-tagged C/EBPα and ε proteins were produced using the TNT Quick Coupled non-denaturing polyacrylamide gel in 0.5x TBE buffer and radioactivity was visualized using the BAS2500 system (Fuji Film). The sequences of oligonucleotide probes were listed in Table S3. ChIP assays and real time PCR were carried out as described previously by using anti-C/EBPε antibody (Santa Cruz) (22,25).
Primers used for the real time PCR were indicated in the Table S4.  Table S6.

RTPCR-
Streptomycin-resistant colonies were cloned and checked to make EBV-BAC C/EBP BSmt.
Electroporation of E. Coli was performed using a Gene Pulser III (Bio-Rad) and purification of EBV-BAC DNA was achieved with NucleoBond Bac100 (Macherey-Nagel).
Recombination was confirmed with PCR products of the promoter region, by electrophoresis of the BamHI-digested viral genome, and sequencing analysis.
EBV-BAC DNA was transfected into HEK293 cells using lipofectamine 2000 reagent (Invitrogen), followed by culture on 10 cm dishes with 100-150 µg/ml of hygromycin B for 10-15 days for cloning of GFP-positive cell colonies as described previously (27).
Briefly, for each recombinant virus, we picked up more than 10 hygromycin-resistant, GFP-positive cell colonies to obtain at least 3 typical clones exhibiting minimal spontaneous expression of viral lytic proteins and significant induction of these upon BZLF1 transfection. handle when compared to other cells featuring type II EBV latency. When C/EBPα or ε were exogenously expressed, increased levels of LMP1 protein were readily detected by immunoblotting (Fig. 1B) while other members did not appreciably increase the LMP1 levels.

Screening of cellular factors transactivating
We also tested C666-1, a nasopharyngeal carcinoma cell line naturally infected with EBV ( Fig. 1C). At least expression of C/EBPα increased LMP1 mRNA level. In AGS-CR2/GFP-EBV-Bac cells, LMP1 is increased by the exogenous supply of not only C/EBPα and ε, but also β. (Fig. S2) Identification of a C/EBP binding site in the LMP1 promoter. Since we confirmed C/EBP's potentiating effect of LMP1 transcription, we then carried out truncation and mutagenesis analysis of the promoter region in order to identify any cis-element that might be responsible for the activation. We first prepared reporter vectors in which the promoter sequences were gradually deleted as shown in Fig. 2A. While truncation of the sequence to -320 relative to the transcription start site (+1) did not impair the promoter response to C/EBPα, severing the sequence between nucleotides -320 and -229 markedly diminished the response (Fig. 2B) We also tried to detect binding of endogenous C/EBP to the LMP1 promoter.
In AGS-CR2/GFP-EBV-Bac cells, C/EBPε was detected on the promoter sequence (Fig. 3B), although C/EBPα was undetectable (not shown). Because amount of C/EBPα is very low in the cell line, we speculate the ChIP result simply reflected the expression level of the family member.
Previous reports demonstrated that a distal promoter, termed TR-L1, located within the terminal repeats (TR) of the viral genome, is also activated in addition to the proximal ED-L1 promoter in certain cell types with EBNA2-independent LMP1 expression (10,13,15,16), we next examined, by RT-PCR, if C/EBP might affect the TR-L1 promoter, too. An antisense primer was designed to jump the first intron of LMP1 gene (Fig. 4A, primer #3), so that the possibility of genomic contamination could be ignored, and one sense primer was set within the first exon (Fig. 4A, primer #1) and another sense set well upstream of the transcription start site (+1) of ED-L1 promoter (Fig. 4A, primer #2). The result of the RTPCR (Fig. 4B) indicated that C/EBPα markedly enhanced transcription from the TR-L1 promoter. It is not clear, from this result, whether the ED-L1 promoter is also activated or not.
Although we already identified the cis-element responsible for the activation of the proximal ED-L1 promoter (Fig. 2), we then searched to find the cis-element that is crucial for the activation of the distal promoter, since the TR-L1 promoter of LMP1 gene was markedly activated by C/EBPα (Fig. 4). We first prepared a firefly luciferase reporter construct by inserting the TR-L1 promoter (nucleotide -1115 to -544, Fig. 5A, TR). Curiously, this reporter did not respond to exogenous expression of C/EBPα (Fig. 5B, TR), suggesting that a functional cis-element responsible for the activation of the TR-L1 promoter does not exist in the sequence between nucleotides -1115 and -544. Therefore, speculating that the C/EBP binding site located within the ED-L1 promoter might act to influence the TR-L1 promoter activity form downstream, the promoter sequence in the reporter construct was extended to -147, to cover the C/EBP motif (Fig. 5A, TR+BS). Although this reporter contains a part of the ED-L1 promoter, transcription from ED-L1 should not initiate since it does not contain the transcription start site (+1) of the ED-L1 promoter. As shown in Fig. 5B (TR+BS), the vector did respond to C/EBPα, and introduction of a point mutation at the C/EBP BS depressed the response (Fig. 5C,  TR+BSmt). In addition, a reporter containing the TR-L1 and complete ED-L1 promoters (Fig. 5A, TR+ED) acted in a similar manner (Fig. 5D, TR+ED, TR+EDmt).
These results suggest that activation of both the TR-L1 and ED-L1 promoters by C/EBP is mediated through the single C/EBP binding site in the ED-L1 promoter. Protein levels were examined in the AGS cells, latently infected with wild-type or mutated EBV (Fig.   7A). Production of LMP1 protein in the AGS cells with virus carrying the point mutation at the C/EBP binding site (Fig. 7A, mt) was obviously lower than in the wild-type. The AGS cells expressed little or no EBNA2, in contrast to LCL (Fig. 7A), indicating that the virus established type II latency in the cells (31). Promoter usage patterns were then checked by RT-PCR using the specific primers used for Fig. 4. Transcription from the TR-L1 promoter was remarkably restricted with the mutant (Fig. 7B), although the effect of the mutation on the ED-L1 promoter was not distinguishable from the data. We also checked that EBNA1 levels were comparable (Fig. 7B).

Mutation in the C/EBP binding site attenuated activity of both LMP1 promoters in
Next, the effects of C/EBP exogenous expression were analyzed in cells carrying recombinant viruses. In AGS cells latently infected with wild-type EBV, intrinsic LMP1 protein was present and ectopic supply of C/EBPα caused prominent increase in LMP1 protein levels (Fig. 7C). On the other hand, in cells with mutant EBV, intrinsic LMP1 protein level was low and C/EBPα expression did not induce increase (Fig. 7C). RT-PCR analysis clearly showed that transcriptional activation of LMP1 gene by C/EBPα in wild-type, at least for the TR-L1 promoter, was diminished in the mutant (Fig. 7D), indicating significance for the motif.

Knockdown of C/EBP reduced LMP1 levels.
Lastly, we tested the effect of endogenous C/EBP proteins on LMP1 expression levels. To this end, α or ε member of C/EBP family was ablated by shRNA technology. In Hela-CR2/GFP-EBV cells, knockdown of either C/EBPα or ε significantly restricted the amount of LMP1 (Fig. 8A, S3). In AGS-CR2/GFP-EBV-Bac cells, we tested knockdown of C/EBPε. Because levels of endogenous C/EBPα in the cells were very low, knockdown of C/EBPα was not done. Treatment of shC/EBPε caused reduction of LMP1 protein in AGS cells, too (Fig. 8B). These results indicate that C/EBP proteins are involved in LMP1 production, and suggest that the effect is dependent on cell types. Subsequent analyses demonstrated that C/EBP enhanced the distal TR-L1 promoter of LMP1, and that the activation was mediated through one C/EBP binding motif in the proximal ED-L1 promoter (Fig. S4).

DISCUSSION
Therefore, the distal TR-L1 promoter is activated by More amazingly, expression patterns of EBV proteins even in type I or type III cells could be modulated by cytokines to resemble those in type II latency (10)(11)(12)36). Hela-CR2/GFP-EBV cells were transfected with empty vector (pcDNA3) or the indicated C/EBP family expression vector.
After 60 h, cell proteins were harvested and subjected to immunoblotting with anti-LMP1, -tubulin, and -Flag antibodies.
(C) C/EBPα transactivated LMP1 levels in a nasopharyngeal carcinoma cell line. C666-1 cells were transfected with empty vector or C/EBPα expression vector. After 48 h, cell RNAs were collected and subjected to RTPCR.