Epstein-Barr virus nuclear antigen 3C interacts with histone deacetylase to repress transcription

EBNA3C can specifically repress the expression of reporter plasmids contain ing EBV Cp latency-associated promoter elements. Cp is normally the main pr omoter for EBNA mRNA initiation, so it appears that EBNA3C contributes to a negative autoregulatory control loop. By mutational analysis it was previo usly established that this repression is consistent with EBNA3C being targe ted to Cp by binding the cellular sequence-specific DNA-binding protein CBF 1 (also known as recombination signal-binding protein [RBP]-J kappa. Furthe r analysis suggested that in vivo a corepressor interacts with EBNA3C in th is DNA binding complex. Results presented here are all consistent with a co mponent of such a corepressor exhibiting histone deacetylase activity. The drug trichostatin A, which specifically inhibits histone deacetylases, reli eved two- to threefold the repression of Cp induced by EBNA3C in two differ ent cell types. Moreover, repression of pTK-CAT-Cp4x by EBNA3C was specific ally enhanced by cotransfection of an expression plasmid for human histone deacetylase-1 (HDAC1). Consistent with these functional assays, in vitro-tr anslated HDAC1 bound to a glutathione S-transferase (GST) fusion protein in cluding full-length EBNA3C, and in the reciprocal experiment EBNA3C bound t o a GST fusion with the N terminus of HDAC1. Coimmunoprecipitations also re vealed an EBNA3C-HDAC1 interaction in vivo, and GST-EBNA3C bound functional histone deacetylase enzyme activity from HeLa cell nuclear extracts. The r egion of EBNA3C involved in the interaction with HDAC1 appears to correspon d to the region which is necessary for binding to CBF1/RBP-J kappa. A direc t physical interaction between EBNA3C and HDAC1 was demonstrated with recom binant proteins purified from bacterial cells, and we therefore conclude th at HDAC1 and CBF1/RBP-J kappa bind to the same or adjacent regions of EBNA3 C, These data suggest that recruitment of histone deacetylase activity make s a significant contribution to the repression of transcription from Cp bec ause EBNA3C bridges an interaction between CBF1/RBP-J kappa and HDAC1.