E2F1, ARID3A/Bright and Oct-2 factors bind to the Epstein-Barr virus C promoter, EBNA1 and oriP, participating in long-distance promoter-enhancer interactions.
The Epstein-Barr virus (EBV) C promoter (Cp) regulates several genes required for B-cell proliferation in latent EBV infection. The family of repeats (FR) region of the latent origin of plasmid replication (oriP) functions as an Epstein-Barr nuclear antigen 1 (EBNA1)-dependent distant enhancer of Cp activity, and the enhancer-promoter interaction is mediated by a higher-order multi-protein complex containing several copies of EBNA1. Using DNA-affinity purification with a 170 bp region of the Cp in combination with mass spectrometry, we identified the cell cycle-regulatory protein E2F1, the E2F-binding protein ARID3A, and the B-cell-specific transcription factor Oct-2 as components of this multi-protein complex. Binding of the three factors to the FR region of oriP was determined by DNA-affinity and immunoblot analysis. Co-immunoprecipitation and proximity ligation analysis revealed that the three factors, E2F1, ARID3A and Oct-2, interact with each other as well as with EBNA1 in the nuclei of EBV-positive cells. Using the chromatin immunoprecipitation assay, we showed that E2F1 and Oct-2 interacted with the FR part of oriP and the Cp, but the ARID3A interaction was, however, only detected at the Cp. Our findings support the hypothesis that EBNA1 initiates transcription at the Cp via interactions between multiple EBNA1 homodimers and cellular transcription factors in a large molecular machinery that forms a dynamic interaction between Cp and FR.
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska University Hospital, University of Gothenburg, SE-41345 Gothenburg, Sweden.
Cecilia
C
Boreström
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska University Hospital, University of Gothenburg, SE-41345 Gothenburg, Sweden.
Alma
A
Forsman
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska University Hospital, University of Gothenburg, SE-41345 Gothenburg, Sweden.
Ulla
U
Rüetschi
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Sahlgrenska University Hospital, University of Gothenburg, SE-41345 Gothenburg, Sweden.
Lars
L
Rymo
D002846:Chromatography, Affinity; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050687:E2F1 Transcription Factor / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D050813:Octamer Transcription Factor-2 / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D025941:Protein Interaction Mapping; D055503:Protein Multimerization; D018741:Replication Origin; D014157:Transcription Factors / Q000378:metabolism*
C490798:ARID3A protein, human; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D019309:Epstein-Barr Virus Nuclear Antigens; D050813:Octamer Transcription Factor-2; D014157:Transcription Factors; C417029:EBV-encoded nuclear antigen 1