Silencing of the Epstein-Barr virus latent membrane protein 1 gene by the Max-Mad1-mSin3A modulator of chromatin structure.

Abstract

The tumor-associated latent membrane protein 1 (LMP1) gene in the Epstein-Barr virus (EBV) genome is activated by EBV-encoded proteins and cellular factors that are part of general signal transduction pathways. As previously demonstrated, the proximal region of the LMP1 promoter regulatory sequence (LRS) contains a negative cis element with a major role in EBNA2-mediated regulation of LMP1 gene expression in B cells. Here, we show that this silencing activity overlaps with a transcriptional enhancer in an LRS sequence that contains an E-box-homologous motif. Mutation of the putative repressor binding site relieved the repression both in a promoter-proximal context and in a complete LRS context, indicating a functional role of the repressor. Gel retardation assays showed that members of the basic helix-loop-helix transcription factor family, including Max, Mad1, USF, E12, and E47, and the corepressor mSin3A bound to the E-box-containing sequence. The enhancer activity correlated with the binding of USF. Moreover, the activity of the LMP1 promoter in reporter constructs was upregulated by overexpression of USF1 and USF2a, and the transactivation was inhibited by the concurrent expression of Max and Mad1. This suggests that Max-Mad1-mediated anchorage of a multiprotein complex including mSin3A and histone deacetylases to the E-box site constitutes the basis for the repression. Removal of acetyl moieties from histones H3 and H4 should result in a chromatin structure that is inaccessible to transcription factors. Accordingly, inhibition of deacetylase activity with trichostatin A induced expression of the endogenous LMP1 gene in EBV-transformed cells.