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

The tumor-associated latent membrane protein 1 (LMP1) gene in the Epstein-B arr virus (EBV) genome is activated by EBV-encoded proteins and cellular fa ctors 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 r egulation of LMP1 gene expression in B cells. Here, we show that this silen cing activity overlaps with a transcriptional enhancer in an LRS sequence t hat contains an E-box-homologous motif Mutation of the putative repressor b inding site relieved the repression both in a promoter-proximal context and in a complete I,RS context, indicating a functional role of the repressor. Gel retardation assays showed that members of the basic helix-loop-helix t ranscription factor family, including Max, Mad1, USF, E12, and E47, and the corepressor mSin3A bound to the E-box-containing sequence. The enhancer ac tivity correlated with the binding of USF. Moreover, the activity of the LM P1 promoter in reporter constructs was upregulated by overexpression of USF 1 and USF2a, and the transactivation was inhibited by the concurrent expres sion of Max and Mad1. This suggests that Max-Mad1-mediated anchorage of a m ultiprotein complex including mSin3A and histone deacetylases to the E-box site constitutes the basis for the repression. Removal of acetyl moieties f rom histones H3 and H4 should result in a chromatin structure that is inacc essible to transcription factors. Accordingly, inhibition of deacetylase ac tivity with trichostatin A induced expression of the endogenous LMP1 gene i n EBV-transformed cells.