Activated mouse Notch1 transactivates Epstein-Barr virus nuclear antigen 2-regulated viral promoters - PubMed (original) (raw)

Activated mouse Notch1 transactivates Epstein-Barr virus nuclear antigen 2-regulated viral promoters

H Höfelmayr et al. J Virol. 1999 Apr.

Abstract

Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for B-cell immortalization by EBV, most probably by its ability to transactivate a number of cellular and viral genes. EBNA2-responsive elements (EBNA2REs) have been identified in several EBNA2-regulated viral promoters, each of them carrying at least one RBP-Jkappa recognition site. RBP-Jkappa recruits EBNA2 to the EBNA2RE and, once complexed to EBNA2, is converted from a repressor into an activator. An activated form of the cellular receptor Notch also interacts with RBP-Jkappa, providing a link between EBNA2 and Notch signalling. To determine whether activated Notch is able to transactivate EBNA2-responsive viral promoters, we performed cotransfection experiments with activated mouse Notch1 (mNotch1-IC) and luciferase constructs of the BamHI C, LMP1, and LMP2A promoters. We present here evidence that mNotch1-IC transactivates viral promoters known to be regulated by EBNA2. As shown for EBNA2, mutations or deletions of the RBP-Jkappa sites diminish or eliminate mNotch1-IC-mediated transactivation of the promoters, pointing to an essential role for Notch-RBP-Jkappa interaction. In addition to RBP-Jkappa, other cellular factors may bind within the EBNA2REs of viral promoters. While some factors appear to play an important role in both EBNA2- and mNotch1-IC-mediated transactivation, others are only important for the activity of either EBNA2 or mNotch1-IC. We could observe specific mNotch1-IC-responsive regions, thereby throwing more light upon which cofactors interact with EBNA2 and mNotch1-IC, thus enabling them to become functionally transactivators in vivo.

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Figures

FIG. 1

Luciferase activity of the RBP-Jκ multimer construct upon titration of mNotch1-IC and EBNA2 expression plasmids. (A) Twenty micrograms of the reporter construct pGa981-16 was cotransfected with 0.1, 1, or 10 μg of the expression plasmids pSG5 mNotch1-IC (black boxes) or pGa986-20 (white boxes) into the EBNA2-negative cell line BL41-P3HR1. The amount of expression plasmid was adjusted to 10 μg with the vector pSG5. The fold transactivation was standardized to the value obtained from cotransfection with 10 μg of the vector control pSG5. Results are averages from two independent experiments. (B) At 24 h after the transfection of 0.1, 1, or 10 μg of pSG5 mNotch1-IC in BL41-P3HR1 cells, the cells were harvested. Equal amounts of protein were loaded onto the gel, and expression of mNotch1-IC was examined by Western blotting with the anti-FLAG antibody. Available antibodies failed to detect EBNA2 at this concentration.

FIG. 1

FIG. 2

The EBNA2RE of the _Bam_HI C promoter can confer mNotch1-IC and EBNA2 responsiveness on either the _Bam_HI C or the β-globin minimal promoter. (A) Schematic representation of the _Bam_HI C promoter luciferase constructs used in the cotransfection assays. In the upper part of the illustration the essential regions of the EBNA2RE of the _Bam_HI C promoter are shown. The two regions interact with RBP-Jκ and CBF2, respectively. In the lower part, the EBNA2REs and mutated sequences of the essential regions are indicated by solid and open boxes, respectively. (B and C) Portions (10 μg) of the expression plasmids pSG5 mNotch1-IC (B) or pGa986-20 (C) were cotransfected with 20 μg of the reporter construct into the EBNA2-negative cell line BL41-P3HR1. The fold transactivation was standardized to the value from cotransfection with 10 μg of the vector control pSG5. The mean values and the standard deviations of four independent experiments are shown.

FIG. 3

The EBNA2RE of the LMP1 promoter can confer mNotch1-IC and EBNA2 responsiveness on either the LMP1 or the β-globin minimal promoter. (A) Schematic representation of the LMP1 promoter luciferase constructs used in the cotransfection assays. In the upper part of the figure the essential regions of the LMP1 promoter EBNA2RE are shown. One sequence element interacts with RBP-Jκ; the other interacts with Spi1. A potential second RBP-Jκ site located beyond the EBNA2RE is indicated. In the lower part the EBNA2REs and mutated sequences of the essential regions are indicated by solid and open boxes, respectively. (B and C) Portions (10 μg) of the expression plasmids pSG5 mNotch1-IC (B) or pGa986-20 (C) were cotransfected with 20 μg of the reporter construct into the EBNA2-negative cell line BL41-P3HR1. The fold transactivation was standardized to the value from cotransfection with the vector control pSG5. The mean values and the standard deviations of four independent experiments are shown.

FIG. 4

The EBNA2RE of the LMP2A promoter can confer mNotch1-IC and EBNA2 responsiveness on the LMP2A minimal promoter. (A) Schematic representation of the LMP2A promoter luciferase constructs used in the cotransfection assays. In the upper part of the figure the essential regions of the EBNA2RE of the LMP2A promoter are shown. Two regions interact with RBP-Jκ, whereas the two others are designated as L2BF2 and L2BF3. In the lower part of the figure the EBNA2REs and mutated sequences of the essential regions are indicated by solid and open boxes, respectively. (B and C) Portions (10 μg) of the expression plasmids pSG5 mNotch1-IC (B) or pGa986-20 (C) were cotransfected with 20 μg of the reporter construct, respectively, into the EBNA2-negative cell line BL41-P3HR1. The fold transactivation was standardized to the value from cotransfection with the vector control pSG5. The mean values and the standard deviations of four independent experiments are shown.

FIG. 5

Analysis of DNA-protein interactions within the EBNA2RE 5′ domain of the LMP2A promoter. An EMSA is shown with nuclear extracts of BL41-P3HR1 cells and of BL41-P3HR1 cells stably transfected with either EBNA2 or mNotch1-IC, which were incubated with the 32P-labelled 54-bp oligonucleotide O54 consisting of positions −209 to −262 relative to the LMP2A RNA start site. To show that EBNA2 or mNotch1-IC is a component of the complex IVE/IVN, the monoclonal anti-EBNA2 antibody R3 or the anti-FLAG monoclonal antibody M2 was added, respectively. Complexes were separated on a 4% polyacrylamide gel. The positions of complexes I, III, IVE, IVN, IVE*, and IVN* are indicated.

FIG. 6

Analysis of DNA-protein interactions within the EBNA2RE 3′ domain of the LMP2A promoter. An EMSA is shown with oligonucleotide O40 consisting of positions −217 to −178 relative to the LMP2A RNA start site as a radioactively labelled probe. The probe was incubated with Raji and HeLa nuclear extracts. For competition, a 5- to 250-fold molar excess of unlabelled oligonucleotide was added to the gel shift reactions. Complexes were separated on a 4% polyacrylamide gel. The positions of complexes A, B, and C are shown.

FIG. 7

Methylation interference analysis of the EBNA2RE 3′ region. The radioactively labelled and randomly methylated oligonucleotide O40 consisting of positions −217 to −178 of the 3′ region was incubated with HeLa nuclear extracts. The protected guanines are shown for complexes A, B, and C in comparison with the free probe. As a control reaction, a Maxam-Gilbert G reaction is shown on the lefthand side. On the righthand side the sequence of the 3′ region from positions −217 to −178 is shown. Protected sequences are marked with asterisks. Minimal extensions of the protection are indicated by boxes.

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Activated mouse Notch1 transactivates Epstein-Barr virus nuclear antigen 2-regulated viral promoters - PubMed (original) (raw)