ABSTRACT
E2F transcription factors are key players in the regulation of proliferation, apoptosis, and differentiation in mammalian cells. E2Fs are negatively regulated by members of the retinoblastoma protein family, Rb, p107 and p130. During adenovirus infection, viral proteins are expressed that displace Rb family members from E2Fs and recruit E2F complexes to viral and cellular promoter regions. This recruitment of E2F involves the induction of stable E2F binding to inverted E2F binding sites in the Ad E2a and cellular E2F-1 promoters and induces both viral and cellular gene expression. The cellular p107 tumor suppressor also displays such regulation of E2F DNA binding activity. p107 induces stable E2F-4/DP binding to inverted E2F binding sites in the Ad E2a and cellular E2F-1 promoters. The induction of E2F DNA binding by p107 minimally requires the sequences in p107 that mediate E2F interaction. The related tumor suppressor, p130, also effects this function. p107 levels increase substantially as cells progress through S phase. p107 induction of E2F DNA binding was observed primarily in S phase cells coincident with the increase in p107 protein levels. The results of promoter activity assays directly correlate the induction of E2F DNA binding by p107 with effective transcriptional repression. These results support a model in which p107 and p130 induce the stable binding of E2F complexes to promoters that drive expression of critical regulatory proteins such as E2F-1. Since p107 and p130 bind histone deacetylase complexes (HDACs) which repress promoter activity, p107-E2F and p130-E2F would stably recruit repressor complexes to effect efficient promoter repression.
Subject(s)
Carrier Proteins , Cell Cycle Proteins , DNA-Binding Proteins , DNA/metabolism , Nuclear Proteins/physiology , Promoter Regions, Genetic , Proteins , Transcription Factors/metabolism , Binding Sites , Cell Line , E2F Transcription Factors , E2F1 Transcription Factor , E2F4 Transcription Factor , Humans , Phosphoproteins/physiology , Retinoblastoma-Binding Protein 1 , Retinoblastoma-Like Protein p107 , Retinoblastoma-Like Protein p130 , S Phase , Transcription Factor DP1 , Transcription Factors/geneticsABSTRACT
The adenovirus type 5 (Ad5) E4-6/7 protein interacts directly with different members of the E2F family and mediates the cooperative and stable binding of E2F to a unique pair of binding sites in the Ad5 E2a promoter region. This induction of E2F DNA binding activity strongly correlates with increased E2a transcription when analyzed using virus infection and transient expression assays. Here we show that while different adenovirus isolates express an E4-6/7 protein that is capable of induction of E2F dimerization and stable DNA binding to the Ad5 E2a promoter region, not all of these viruses carry the inverted E2F binding site targets in their E2a promoter regions. The Ad12 and Ad40 E2a promoter regions bind E2F via a single binding site. However, these promoters bind adenovirus-induced (dimerized) E2F very weakly. The Ad3 E2a promoter region binds E2F very poorly, even via a single binding site. A possible explanation of these results is that the Ad E4-6/7 protein evolved to induce cellular gene expression. Consistent with this notion, we show that infection with different adenovirus isolates induces the binding of E2F to an inverted configuration of binding sites present in the cellular E2F-1 promoter. Transient expression of the E4-6/7 protein alone in uninfected cells is sufficient to induce transactivation of the E2F-1 promoter linked to chloramphenicol acetyltransferase or green fluorescent protein reporter genes. Further, expression of the E4-6/7 protein in the context of adenovirus infection induces E2F-1 protein accumulation. Thus, the induction of E2F binding to the E2F-1 promoter by the E4-6/7 protein observed in vitro correlates with transactivation of E2F-1 promoter activity in vivo. These results suggest that adenovirus has evolved two distinct mechanisms to induce the expression of the E2F-1 gene. The E1A proteins displace repressors of E2F activity (the Rb family members) and thus relieve E2F-1 promoter repression; the E4-6/7 protein complements this function by stably recruiting active E2F to the E2F-1 promoter to transactivate expression.
Subject(s)
Adenoviridae/metabolism , Adenovirus E4 Proteins/pharmacology , Carrier Proteins , Cell Cycle Proteins , Promoter Regions, Genetic , Transcription Factors/genetics , Adenoviridae/genetics , Amino Acid Sequence , Binding Sites , Cell Line , Cloning, Molecular , DNA/metabolism , DNA-Binding Proteins/genetics , E2F Transcription Factors , E2F1 Transcription Factor , Electrophoresis, Agar Gel , Gene Expression Regulation, Viral/drug effects , HeLa Cells , Humans , Immunohistochemistry , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Retinoblastoma-Binding Protein 1 , Sequence Alignment , Transcription Factor DP1 , Transcription Factors/analysis , Transcription Factors/biosynthesisABSTRACT
Three regulatory regions on the promoter of CHO adenine phosphoribosyltransferase gene (APRT) were identified. Spacing constraints between these regions were analyzed. With normal spacing, region II (-33 to +19), which separates regions I (-101 to -53) and III (+56 to +85), is critical for APRT transcription. However, when regions I and III are artificially placed in proximity to each other (region II deleted), they are able to drive transcription as efficiently as the wild-type APRT promoter. Neither region I nor III alone is sufficient for efficient transcription. As the spacing between the two regions increases, the transcription decreases. Region I may activate transcription in two ways: through a stringent sequence-specific manner (as in the transcription mediated by regions I and III) and through a manner with relaxed requirement for sequence specificity (as in the transcription from the wild-type APRT promoter).
