Epstein-Barr virus nuclear antigen 3C is a powerful repressor of transcription when tethered to DNA

Abstract

The expression of Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for the activation and immortalization of human B lymphocytes by EBV. EBNA3C consists of 992 amino acids and includes a potential bZIP motif and regions rich in acidic, proline, and glutamine residues. Thus, EBNA3C resembles several trans regulators of gene expression. It has recently been shown that a fragment of EBNA3C can activate reporter gene expression when fused to the DNA-binding domain of GAL4 (D. Marshall and C. Sample, J. Virol. 69:3624-3630, 1995). Although EBNA3C binds DNA, a specific site for EBNA3C binding has not been identified; to test the ability of full-length EBNA3C to regulate transcription, EBNA3C (amino acids 11 to 992) was fused to the DNA-binding domain of GAL4. We show that this fusion protein does not transactivate but rather is a potent repressor of reporter gene expression. Repression is dependent on the dose of GAL4-EBNA3C and on the presence of GAL4-binding sites within reporter plasmids. Repression is not restricted to B cells nor is it species or promoter specific. Repression is independent of the location of the GAL4-binding sites relative to the transcription start site. A fragment of EBNA3C (amino acids 280 to 525) which represses expression in a manner which is nearly identical to that of the full-length protein has been identified; this fragment is rich in acidic and proline residues. A second, less potent repressor region located C terminal to amino acids 280 to 525 has also been identified; this domain is rich in proline and glutamine residues. We also show binding of EBNA3C, in vitro, to the TATA-binding protein component of TFIID, and this suggests a mechanism by which EBNA3C may communicate with the basal transcription complex.

Epstein-Barr Virus Nuclear Antigen 3C Interacts with Histone Deacetylase To Repress Transcription

“…Cell debris was removed by centrifugation (at 11,600 ϫ g for 20 min at RT), and the supernatant was transferred to a fresh Eppendorf tube for a CAT or luciferase assay. Luciferase and CAT assays have been described previously (7,37). All values were normalized to ␤-galactosidase activity expressed from 2 g of pSV-␤-Gal plasmid, which was included as an internal control for transfection efficiency and any cytotoxic effects of the tested DNA.…”

“…Expression of GST fusion proteins in Escherichia coli. The expression of fusion proteins was performed basically as described previously (7). Briefly, 50 ml of an overnight culture was inoculated into 500 ml of 2ϫ TY medium containing 50 g of ampicillin/ml.…”

“…It has been suggested that EBNA3C binding to CBF1/RBP-J prevents the association of EBNA2-CBF1/RBP-J complexes with DNA and that in this way EBNA3C represses the EBNA2-mediated activation of various natural and synthetic promoters, which include CBF1/RBP-J sites (19,41,48,57). However, fusions of EBNA3C with the DNA binding domain of the Saccharomyces cerevisiae transactivator GAL4 have shown that, when tethered to DNA, EBNA3C is a very potent repressor of reporter gene transcription in its own right (7,48). Subsequently, we also showed that EBNA3C specifically represses Cp, the major promoter for EBNA expression in LCLs.…”

Viral Latency and Its Regulation: Lessons from the γ-Herpesviruses

“…This switch is driven by the interaction of EBNA2 with the cellular transcription factor RBP-Jk (also known as CBP-1 and CSL), which normally functions in the regulation of genes in the cellular Notch signaling pathway (Grossman et al, 1994;Henkel et al, 1994;Waltzer et al, 1995;Zimber-Strobl et al, 1994). Two other EBNAs, EBNA3A and EBNA3C, also interact with RBP-Jk and displace EBNA2 -thus serving as modulators of EBNA2's transcriptional regulatory activity (Bain et al, 1996;Johannsen et al, 1996;Radkov et al, 1997;Radkov et al, 1999;Robertson et al, 1995;Waltzer et al, 1996;Zhao et al, 1996). EBNA2 interaction with RBP-Jk also serves to turn on transcription of the LMP genes.…”

Epstein-Barr virus immortalization: Notch2 interacts with CBF1 and blocks differentiation

“…EBNA2 binds to the repression domain of CBF1, blocking repression function, and relief of repression combined with the presence of the strong EBNA2 transcriptional activation domain transactivates expression of genes containing upstream CBF1 binding sites (24). Two of the other essential immortalizing proteins, EBNA-3A and EBNA-3C, also bind to CBF1 (4,52,53,69). The interaction domain on CBF1 for these proteins lies amino terminal to that for EBNA2.…”