Epstein-Barr virus nuclear antigen 3C interacts with histone deacetylase to repress transcription.
EBNA3C can specifically repress the expression of reporter plasmids containing EBV Cp latency-associated promoter elements. Cp is normally the main promoter for EBNA mRNA initiation, so it appears that EBNA3C contributes to a negative autoregulatory control loop. By mutational analysis it was previously established that this repression is consistent with EBNA3C being targeted to Cp by binding the cellular sequence-specific DNA-binding protein CBF1 (also known as recombination signal-binding protein [RBP]-Jkappa. Further analysis suggested that in vivo a corepressor interacts with EBNA3C in this DNA binding complex. Results presented here are all consistent with a component of such a corepressor exhibiting histone deacetylase activity. The drug trichostatin A, which specifically inhibits histone deacetylases, relieved two- to threefold the repression of Cp induced by EBNA3C in two different cell types. Moreover, repression of pTK-CAT-Cp4x by EBNA3C was specifically enhanced by cotransfection of an expression plasmid for human histone deacetylase-1 (HDAC1). Consistent with these functional assays, in vitro-translated HDAC1 bound to a glutathione S-transferase (GST) fusion protein including full-length EBNA3C, and in the reciprocal experiment EBNA3C bound to a GST fusion with the N terminus of HDAC1. Coimmunoprecipitations also revealed an EBNA3C-HDAC1 interaction in vivo, and GST-EBNA3C bound functional histone deacetylase enzyme activity from HeLa cell nuclear extracts. The region of EBNA3C involved in the interaction with HDAC1 appears to correspond to the region which is necessary for binding to CBF1/RBP-Jkappa. A direct physical interaction between EBNA3C and HDAC1 was demonstrated with recombinant proteins purified from bacterial cells, and we therefore conclude that HDAC1 and CBF1/RBP-Jkappa bind to the same or adjacent regions of EBNA3C. These data suggest that recruitment of histone deacetylase activity makes a significant contribution to the repression of transcription from Cp because EBNA3C bridges an interaction between CBF1/RBP-Jkappa and HDAC1.
Radkov
S A
SA
Section of Virology and Cell Biology, Imperial College of Science, Technology and Medicine, St Mary's Campus, London W2 1PG, United Kingdom.
Touitou
R
R
Brehm
A
A
Rowe
M
M
West
M
M
Kouzarides
T
T
Allday
M J
MJ
D000818:Animals; D001665:Binding Sites; D002457:Cell Extracts; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D019169:Jurkat Cells; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D019309:Epstein-Barr Virus Nuclear Antigens; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D009687:Nuclear Proteins; C497570:RBPJ protein, human; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C012589:trichostatin A; D005982:Glutathione Transferase; D006655:Histone Deacetylases