Activation of the Notch-regulated transcription factor CBF1/RBP-Jkappa through the 13SE1A oncoprotein

Abstract

Signaling through the Notch pathway controls cell growth and differentiation in metazoans. Following binding of its ligands, the intracellular part of the cell surface Notch1 receptor (Notch1-IC) is released and translocates to the nucleus, where it alters the function of the DNA-binding transcription factor CBF1/RBP-Jkappa. As a result, CBF1/RBP-Jkappa is converted from a repressor to an activator of gene transcription. Similarly, the Epstein Barr viral oncoprotein EBNA2, which is required for B-cell immortalization, activates genes through CBF1. Moreover, the TAN-1 and int-3 oncogenes represent activated versions of Notch1 and Notch4, respectively. Here, we show that the adenoviral oncoprotein 13S E1A also binds to CBF1/RBP-Jkappa, displaces associated corepressor complexes, and activates CBF1/RBP-Jkappa-dependent gene expression. Our results suggest that the central role of the Notch-CBF1/RBP-Jkappa signaling pathway in cell fate decisions renders it susceptible to pathways of viral replication and oncogenic conversion.

Figure 1

13SE1A activates gene transcription via CBF1. (A) Murine HES1 promoter reporter construct (black bars) or a promoter construct that carries a mutation in the HES1–CBF1 binding site (white bars) was transfected in HD3 cells together with genomic E1A, 13SE1A, 12SE1A, or Notch1-IC (N-IC), as indicated. (B) Epstein-Barr viral LMP2A promoter (black bars) or a construct with a deletion encompassing the 54 bp LMP2A–CBF1 response element (white bars) was transfected together with E1A or Notch1-IC as in A. (C, left) Artificial CBF1 responsive promoters that carry one (black bars), six wild-type (grey bars), or one mutated LMP2A–CBF1 response element (white bars), respectively, fused to the minimal β-globin core promoter (hatched bars), were transfected together with E1A or Notch1-IC as in A. (Right) Reporter activation of the same constructs in the 293 cells that carries a chromosomally integrated single copy of genomic E1A. (D) CBF1 was fused to the c-Myb DNA-binding domain (DBD) and cotransfected with 13S E1A in HD3 cells. Activation of reporter expression from a Myb-responsive promoter was determined following transfection of effector plasmid combinations as indicated.

Figure 2

13SE1A binds to CBF1 and releases an associated corepressor complex. (A) QT6 fibroblasts were transfected with E1A and/or Flag-tagged CBF1 cDNA expression vectors as indicated. Cellular protein complexes were immunoprecipitated with monoclonal E1A antibody. Coimmunoprecipitated (CoIP) and ectopic expression of CBF1 were revealed by immunoblotting with Flag M2 antibody. (B) E1A interacts with endogenous CBF1. Protein extracts from 293 cells were immunoprecipitated with anti-c-myc or anti-E1A antibodies as indicated. CoIP CBF1 was revealed by immunoblotting using anti-CBF1. Fibroblasts transfected with the murine CBF1 protein were used as control (lane 1). (C) Various in vitro translated CBF1 fragments (left) were subjected to binding to GST or GST–13SE1A fusion proteins as indicated on the top. (D) SMRT abolishes 13SE1A mediated transactivation via CBF1. Reporter expression from the hexamerized LMP2A–CBF1 reporter construct was determined as described in Fig. 1C. A limiting amount of 13SE1A effector plasmid (50 ng) was transfected. SMRT effector plasmid concentrations were 0.1, 0.4, and 0.8 μg, respectively. The ratio between reporter activity obtained with the hexamerized CBF1 response element in relation to the core promoter is shown. (E) Interaction between E1A and CBF1 is inhibited by SMRT. QT6 fibroblasts were transfected with expression vectors encoding Flag-tagged CBF1 (3 μg), 13S E1A (3 μg), and SMRT (3 and 10 μg), as indicated. Immunoprecipitation and CBF1 detection were performed as described in A.

Figure 3

E1A binding and CBF1 activity. (A) Schematic representation of E1A proteins indicating conserved regions (CR) and amino acid numbers on the top. (B) Binding of radiolabeled E1A proteins as depicted on the left (lane 1; input, 10% of reaction) to GST (lane 2; specificity control) or GST–CBF1 fusion protein (lane 3) comprising the N-terminal 205 amino acids of CBF1. (C) Reporter expression from the β-globin-promoter with 6 LMP2A–CBF1 response elements (as in Fig. 1C and 2D). (D) Activation of a Myb–CBF1 fusion protein by E1A was examined on a Myb responsive promoter, similarly to Fig. 1D. Reporter activation through Myb–CBF1 (or MybDBD as a control) in presence of E1A proteins was examined. Ratio of reporter activities in presence of Myb–CBF1 versus MybDBD is represented.

Figure 4

CR3 C-terminal and N-terminal sequences are required for CBF1 activation. (A, left) Activation of the murine HES1 promoter reporter construct (as in Fig. 1A). (Right) Activation of the Myb–CBF1 fusion protein (as in Fig. 1D) through E1A mutants. Ratio of reporter activities in presence of Myb–CBF1 versus MybDBD is represented. (B) CR3 C-terminal sequence binds to CBF1. QT6 fibroblasts were transfected with Flag-tagged CBF1 and E1A cDNA expression vectors as indicated. Immunoprecipitation and CBF1 detection were performed as described in Fig. 2A. (C) Functional replacement of the CBF1 interacting RAM domain of Notch-IC by E1A CR3. Schematic representation of the constructs used in D and E. (N-IC) Notch-IC; (N-ΔRAM) Notch-IC deleted of its RAM domain; (E1A-N-IC) the RAM domain of Notch has been replaced by CR3 (residues 140–204 or 177–204). (D) Reporter expression from 6 LMP2A–CBF1 response elements, as in Fig. 2C. (E) Reporter expression from the murine HES1 promoter reporter construct or from a promoter construct that carries a mutation in the CBF1 binding site, as in Fig. 1A. The ratio between wild type versus mutant promoter is shown.