The ErbB3 binding protein Ebp1 interacts with Sin3A to repress E2F1 and AR-mediated transcription

Ectopic expression of ebp1, a member of the PA2G4 family, inhibits the proliferation and induces the differentiation of human breast and prostate cancer cell lines. Ebp1 inhibits transcription of E2F1 and androgen receptor regulated genes such as prostate specific antigen (PSA) through its interactions with histone deacetylases (HDACs). To further understand Ebp1's interactions with other components of the transcriptional repression machinery, we examined the association of Ebp1 with the corepressor Sin3A. Ebp1 interacted with Sin3A both in vitro and in vivo as demonstrated by glutathione S-transferase (GST) pull-down and coimmunoprecipitation analysis. The C-terminal domain of Ebp1, responsible for its ability to repress transcription and arrest cell growth, was necessary and sufficient for binding Sin3A. The C-terminal domain of Sin3A, containing the paired amphipathic domain 4 and the HDAC interacting domain, bound Ebp1. Recombinant Sin3A bound Ebp1 directly, but recombinant HDAC2 failed to bind Ebp1. Chromatin immunoprecipitation (ChIP) and DNA affinity precipitation analysis demonstrated that Ebp1 and Sin3A associate at the PSA and E2F1 promoters. Functionally, Sin3A enhanced the ability of Ebp1 to repress transcription of androgen receptor (AR) and E2F1 regulated genes. These results demonstrate that Ebp1 participates in transcriptional regulation via its interaction with the Sin3–HDAC

The N-Terminal 24 Amino Acids of the p55 Gamma Regulatory Subunit of Phosphoinositide 3-Kinase Binds Rb and Induces Cell Cycle Arrest

Although phosphoinositide 3-kinase (PI 3-kinase) is essential for cell cycle progression, the molecular mechanisms that regulate its diverse biological effects are poorly understood. We demonstrate here that Rb, a key regulator of cell cycle progression, associates with p55 kDa (p55α and p55γ) regulatory subunits of PI 3-kinase in vivo and in vitro. Both confocal microscopy and biochemical analysis demonstrated the presence of p55γ in the nucleus. The 24-amino-acid N-terminal end of p55γ, which is unique among PI 3-kinase regulatory subunits, was sufficient to bind Rb. Addition of serum or growth factors to quiescent cells triggered the dissociation of Rb from p55. Ectopic expression of the 24-amino-acid N-terminal end of p55γ inhibited cell cycle progression, as evidenced by induction of cell growth arrest at the G(0)/G(1) phase, inhibition of DNA synthesis, inhibition of cyclin D and cyclin E promoter activity, and changes in the expression of cell cycle-related proteins. The inhibitory effects of the N-terminal end of p55γ on cell cycle progression depended on the presence of functional Rb. These data demonstrate for the first time an association of p55γ with Rb and show that modification of this association can lead to cell cycle arrest

Endogenous Ebp1 and Sin3A bind to an E2F1 oligonucleotide MCF-7 cells were transfected with a control EGFP vector (GFP) or an EGFP–Ebp1 (Ebp1) vector

<p><b>Copyright information:</b></p><p>Taken from "The ErbB3 binding protein Ebp1 interacts with Sin3A to repress E2F1 and AR-mediated transcription"</p><p>Nucleic Acids Research 2005;33(18):6024-6033.</p><p>Published online 27 Oct 2005</p><p>PMCID:PMC1270947.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Transfected cells were selected in 500 µg/ml of G-418 for 1 week and collected as described. Untransfected logarithmically growing MCF-7 cells were also lysed (Con). Cell lysates were incubated with a biotinylated double-stranded E2F1 consensus oligonucleotide. DNA bound proteins were precipitated by magnetic streptavidin beads. Western blotting was used to detect the presence of bound proteins. Input is illustrated in A and proteins associated with the E2F1 oligo in B. The GFP antibody detected the presence of exogenous Ebp1 (upper panel); a polyclonal antibody to Ebp1 detected the presence of endogenous Ebp1 (middle panel); a mouse monoclonal antibody to Sin3A detected endogenous Sin3A (lower panel)

Assembly of Ebp1, Sin3A and HDAC2 complexes at the promoter of the PSA gene in response to bicalutamide treatment

<p><b>Copyright information:</b></p><p>Taken from "The ErbB3 binding protein Ebp1 interacts with Sin3A to repress E2F1 and AR-mediated transcription"</p><p>Nucleic Acids Research 2005;33(18):6024-6033.</p><p>Published online 27 Oct 2005</p><p>PMCID:PMC1270947.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Schematic diagram of the PSA gene regulatory region. ( and ) ChIP assays of Ebp1 involvment in the PSA gene regulatory region. Log phase LNCaP cells, in the absence or presence of bicalutamide as indicated, were fixed with formaldehyde and chromatin lysates immunoprecipitated with pre-immune IgG or antibodies to Ebp1, Sin3A or HDAC2 as indicated. Samples were processed as described in the Materials and Methods and PCR products visualized by ethidium bromide staining. Immunoprecipitated DNA was amplified by using primers specific for the human PSA promoter ARE I or ARE II as illustrated in (A). IN = Input which represents 1% of the total amount of chromatin added to each immunoprecipitation reaction. M = MW markers. Diamond headed arrow indicates the position of the 211 bp PCR product. In (C) cells were treated with bicalutamide