Effects of mutations in the UP site on the binding of recombinant GABP alpha/beta

<p><b>Copyright information:</b></p><p>Taken from "Characterization of a negative transcriptional element in the promoter"</p><p>http://breast-cancer-research.com/content/9/4/R49</p><p>Breast cancer research : BCR 2007;9(4):R49-R49.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC2206725.</p><p></p> Bandshift reactions were performed with recombinant human GABP alpha and beta proteins and P-labelled wild-type (UPFR6) and mutant UPFR6 site probes, as described in Figure 4a with the probes described in Figure 3b. Complexes are as indicated

Effects of mutations in the UP site on the binding of endogenous nuclear proteins

<p><b>Copyright information:</b></p><p>Taken from "Characterization of a negative transcriptional element in the promoter"</p><p>http://breast-cancer-research.com/content/9/4/R49</p><p>Breast cancer research : BCR 2007;9(4):R49-R49.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC2206725.</p><p></p> The sequence of the proximal promoter is shown with previously characterized sites boxed. E2F sites characterized by Bindra and Glazer [27] (E2FA and E2FB) are shown by light grey boxes while the first exon is shown by the darker box and by the arrow showing the transcriptional start site. The arrows indicate the potential /GABP binding sites. The sequence of the UPFR6 probe is indicated, which encompasses both the UP and E2F sites (dotted and grey box marked E2FB). Arrows identify putative GABP alpha/beta binding sites. The sequences of the various mutant probes are indicated along with their names. The P-labelled UPFR6 probes described in (b) were incubated in binding reactions with 5 μg of nuclear extract from the MCF-7 and T-47D cell lines and bandshift assays were performed. Only the DNA/protein complexes are shown, with the Upper, Lower and non-specific (NS) bands indicated as in Figure 1

Binding of recombinant, nuclear extract derived and endogenous GABP alpha/beta to the promoter

<p><b>Copyright information:</b></p><p>Taken from "Characterization of a negative transcriptional element in the promoter"</p><p>http://breast-cancer-research.com/content/9/4/R49</p><p>Breast cancer research : BCR 2007;9(4):R49-R49.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC2206725.</p><p></p> A bandshift scanning assay of the promoter for GABP complex binding sites was preformed using recombinant GABP alpha/beta. Double-stranded DNA probes spanning putative protein binding sites were designed for the entire length of the proximal promoter and are indicated on the schematic (top). Binding reactions were performed with recombinant human GABP alpha and beta proteins and each of the P-labelled probes as indicated by the vertical lines. Samples were run on a 6% nondenaturing acrylamide gel and visualized by autoradiography. The locations of the free and bound probe are indicated. Supershift assays with the UPFR6 probe were performed with 5 μg of MCF-7 nuclear extract and an antibody directed against CREB, GABP alpha or Ets-2. Complexes are as indicated. ChIP assays were preformed using chromatin isolated from MCF-7 cells with antibodies against acetylated histone H3, GABP beta, GABP alpha and haemagglutinin (HA). PCR products obtained using -specific primers and the immunoprecipitation products are shown

Cotransfection of the GABP alpha/beta expression vectors and the GABP alpha shRNA with RIBS and UP site multimer reporter constructs

<p><b>Copyright information:</b></p><p>Taken from "Characterization of a negative transcriptional element in the promoter"</p><p>http://breast-cancer-research.com/content/9/4/R49</p><p>Breast cancer research : BCR 2007;9(4):R49-R49.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC2206725.</p><p></p> MCF-7 and T-47D cells were transiently transfected with 125 ng of GF-TATA-renilla-luciferase reporter containing oligomerised RIBS or UP sites (RIBSn-pRL, UPn-pRL), 25 ng of internal control vector (CMV-Luc), and either 25 ng each of GABP alpha and beta expression constructs (light grey bars), or 50 ng of their empty control vector (pCAGGs; white bars), or 50 ng of GABP alpha shRNA (black bars) or its empty control vector (H1–2; dark grey bars). Cells were harvested 48 hours post-transfection and analysed for luciferase activity. The data presented are the mean values ± standard deviation of a representative experiment performed in triplicate and normalized as described in Figure 2. Statistical significance was determined using a -test and significant results (p = 0.05) are indicated by asterisks and are in relation to the empty vector control for each condition (pCAGGS or H1–2)

Model of GABP alpha/beta interaction with the UP site and its effect on promoter activity

<p><b>Copyright information:</b></p><p>Taken from "Characterization of a negative transcriptional element in the promoter"</p><p>http://breast-cancer-research.com/content/9/4/R49</p><p>Breast cancer research : BCR 2007;9(4):R49-R49.</p><p>Published online 30 Jul 2007</p><p>PMCID:PMC2206725.</p><p></p> Schematic representations of the regulation of the promoter. In both MCF-7 and T-47D cells (upper diagram) the RIBS and UP sites, respectively, are shown to bind GABP alpha/beta (shaded circles represent the DNA binding alpha subunit, with the beta subunit indicated by the dark line) while the E2F site has previously been suggested to bind E2F1, E2F4 and E2F6. The UP site also binds another unknown protein (oval) and together the UP and E2F sites are able to recruit an unknown co-repressor. The GA mutant of the UP site (x) disrupts binding of GABP alpha/beta while the US mutant affects the binding of the other unidentified factor. In T-47D cells this other factor does not appear to be functional (dashed oval). See text for explanation