Repression of p53-mediated Transcription by Adenovirus E1B 55-kDa Does Not Require Corepressor mSin3A and Histone Deacetylases

Lisa Y Zhao,Aleixo Santiago,Jilin Liu,Daiqing Liao

doi:10.1074/jbc.m610749200

Abstract

The Ad E1B 55-kDa protein (E1B) is a potent transcriptional repressor. In vitro biochemical studies revealed that direct p53-E1B interaction is essential for E1B to block p53-activated transcription and a corepressor may be involved. To understand how E1B represses p53-mediated transcription in vivo, we expressed E1B in several tumor cell lines that express wild type p53. Here we show that E1B strongly suppresses the expression of p53 target genes such as p21 and Puma-alpha in normal growth conditions or after cells were treated with p53-activating chemotherapeutic agents, suggesting that E1B-mediated gene repression is dominant and cannot be reversed via p53 activation. Interestingly, we found that E1B binds to corepressor mSin3A. Mutagenesis analysis indicated that the sequence motif "LHLLA" near the NH(2) terminus of E1B is responsible for mSin3A binding, and this motif is conserved among E1B proteins from different Ad serotypes. The conserved paired amphipathic helix domain 1 of mSin3A is critical for mSin3A-E1B interaction. Surprisingly, E1B mutants that cannot bind to mSin3A can still repress p53 target genes, indicating that it is not the corepressor required for E1B-mediated gene repression. In support of this notion, repression of p53 target genes by E1B is insensitive to HDAC inhibitor trichostatin A. We further show that both the NH(2)- and COOH-terminal domains of E1B are required for the repression function. Therefore, E1B employs a unique repression mechanism to block p53-mediated transcription.

Highlights

The p53 and pRb tumor suppressor pathways are inactivated in virtually all human cancers regardless of their etiology [1, 2]
To assess whether E1B can repress p53-dependent transcription, the parental LN-229 cells or the transduced cells were mock treated with vehicle (Me2SO), the HDAC inhibitor trichostatin A (TSA), chemotherapeutic agent 5-FU, or a combination of TSA and 5-FU
This study provides some important insights into the mechanism by which E1B represses transcription

Summary

The abbreviations used are

Adenovirus; 5-FU, 5-fluorouracil; aa, amino acid; Ad12, adenovirus type 12; CBP, CREB-binding protein; ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; GFP, green fluorescent protein; HDAC, histone deacetylases; IP, immunoprecipitation; ORF, open reading frame; PAH, paired amphipathic helix domain; PIC, transcriptional preinitiation complex; SD, synthetic dropout medium; TAD, transactivation domain; TBP, TATA-binding protein; WT, wild type; PIPES, 1,4piperazinediethanesulfonic acid; TSA, trichostatin A. Besides interactions with the aforementioned coactivators, the TAD of p53 interacts with components of basal transcriptional machinery or the Mediator complex (for a recent review see Ref. 17). These contacts stimulate the recruitments of TFIID and TFIIA, and stabilize the preinitiation complex (PIC) assembled on the DNA template containing p53-binding sites [17], which contributes to p53-mediated transactivation. We report here that E1B strongly suppresses the expression of p21 and Puma-␣, two prototypical p53 target genes This repression is not affected by p53-activating agents, which contrasts with the observed alleviation of MDM2-mediated inhibition of p53 upon DNA damage-induced phosphorylation [21]. These findings suggest a unique mechanism of gene repression by E1B

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION