The BT474 cell line was maintained at 37°C in a humidified atmosphere of 5% CO₂ in air. Cell lines were routinely cultured in RPMI-1640 media supplemented with 10% FBS (Sigma, St. Louis, MO). The T47D EBP1-silenced and BT474 EBP1-overexpressing cell lines have been previously described (21).

The ErbB2 promoter reporter plasmid encoding the -500 bp proximal ErbB2 promoter was a gift from Dr Chris Benz (University of California, San Francisco) (22). The -3798 and -6007 bp plasmids encoding distal promoters described by Delacroix et al(23) were a gift from Dr Jean Imbert (Université de la Méditerranée) (24). Wild-type and mutant EBP1 expression plasmids used in this study were previously described (17).

The method demonstrated by Shang et al(25) was used. Briefly, BT474 cells (3–4×10⁶) were grown in 150-mm dishes in RPMI-1640 medium supplemented with 10% FBS. After 24 h of culture, cells were transferred to serum-free RPMI-1640 media overnight. Cells were then left untreated or treated with 50 ng/ml of HRGβ1 (R&D Systems, Minneapolis, MN) for 20 min. Formaldehyde (1%) was added for 10 min and the reaction was quenched using 0.125 M glycine. Cells were harvested, pelleted and the pellets were resuspended in 0.3 ml of lysis buffer [1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1) and 0.5% NP-40] and 1X protease inhibitor cocktail (Roche, Indianapolis, IN). Chromatin was sheared to an average size of 500 bp by sonication using a Bioruptor ultrasonicator (Diagenode, Sparta, NJ) and diluted in ChIP dilution buffer (Millipore, Billerica, MA) to a final volume of 1 ml. A portion of the diluted cell supernatant (1%) was used to quantify the amount of DNA present in the samples. The EBP1 antibody (2 μg/reaction mixture) (Millipore) or negative ChIP validated pre-immune IgG (Sigma) as a control was added to the samples. ChIP validated agarose beads (Millipore) were added for another 6 h. Agarose-bead complexes were washed sequentially in low salt, high salt, LiCl and TE buffer provided in a kit from Millipore and extracted 2 times with freshly prepared elution buffer (1% SDS, 0.1 M NaHCO₃). Eluates were pooled and incubated at 65°C for 6 h to reverse the formaldehyde cross-linking. DNA was purified by phenol-chloroform extraction and precipitated in the presence of 0.3 M sodium acetate and 1 μg glycogen in 2 volumes of ethanol at −20°C overnight. The DNA pellets were dissolved in 5 μl of sterile water. The gene-specific primer sequences were: -500 bp F, 5′-GGG GTC CTG GAA GCC ACA AGG TAA-3′ and R, 5′-ACT TTC CTG GGG AGC TTG CAT CCT-3′; -4600 bp F, 5′-TCC CCA GCA ACC TGT GCC TCA-3′ and R, 5′-ACC AGC CAG CTT GGG GTC AGA-3′; GAPDH F, 5′-ATG GTT GCC ACT GGG GAT CT-3′ and R, 5′-TGC CAA AGC CTA GGG GAA GA-3′. The MyiQ real-time PCR detection system and SYBR-Green PCR mix (Bio-Rad, Richmond, CA) were used to perform real-time PCR. The relative quantitation of targeted genes was determined by the comparative ΔΔCt (threshold) method using GAPDH as an internal control (14). Known quantities of input DNA were used to quantify the PCR products. In individual experiments, 3 wells were set up per data point. The data shown are the means ± SE from 3 independent experiments.

For the ErbB2 reporter assays, BT474 cells (1×10⁵) were plated in 6-well plates in complete media. When cells reached 50–60% confluency, they were transfected with 1 μg of an ErbB2 reporter plasmid and 5 ng of Renilla (pRL)-TK control plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Cell lysates were collected 48 h later and luciferase activity was assessed using a Promega Dual-Luciferase assay kit (Madison, WI). All transfection experiments were performed in triplicate wells and repeated 3 times. The activities of Renilla luciferase were used to normalize any variations in transfection efficiency. The data are expressed as relative light units (RLU) which is the ratio of ErbB2-luc RLU:pRL-TK RLU for each sample.

Total cell extracts were prepared by direct lysis of cells with lysis buffer [50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 250 mM NaCl, 1% Triton X-100, 0.5 mM DTT and 1X Complete™ protease inhibitor]. Protein concentrations were measured using a detergent compatible kit (Bio-Rad). Proteins (30 μg/well) were resolved by SDS-PAGE, transferred to PVDF membranes and immunoblotted as previously described (2). The EBP1 antibody was obtained from Millipore and the anti-actin FLAG and GAPDH antibodies were from Sigma.

For immunoprecipitation (IP) of endogenous ErbB2 mRNA-EBP1 protein complexes (RNP), cell lysates (1.5 mg) were incubated for 2 h at 4°C with protein A-Sepharose beads (Calbiochem) that had been precoated with 3 μg of pre-immune IgG (Sigma) or antibodies recognizing EBP1 or HuR (Santa Cruz Biotechnology, Santa Cruz, CA). Beads were washed with NT2 buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM MgCl₂ and 0.05% NP-40], incubated with 20 units of RNase-free DNase I (15 min, 30°C), followed by incubation with 100 μl NT2 buffer containing 0.1% SDS and 0.5 mg/ml proteinase K for another 30 min at 55°C. The RNA isolated from the IP was converted to cDNA using gene-specific primer pairs F, 5′-GGGAAGAATGGGGTCGTCAAA-3′ and R, 5′-CTCCTCCCTGGGGTGTCAAG-3′ and amplified by real-time quantitative PCR as described.

Cells were serum-starved overnight and then treated with actinomycin D (5 μg/ml). Cells were harvested at sequential time points following actinomycin addition. Total RNA was extracted with Trizol and DNase treated for RT-qPCR analysis. The data represent the means ± SE of three to five independent experiments.

Results were analyzed using a two-tailed Student’s t-test. A value of P<0.05 was considered to indicate a statistically significant difference.

We previously showed that ectopic expression of EBP1 decreased the activity of an ErbB2 luciferase reporter that encodes the 500-bp proximal promoter upstream from the transcription start site (21). We, therefore, aimed to determine the effect of the inhibition of EBP1 expression on ErbB2 promoter activity. T47D cells were used in which EBP1 expression was silenced by shRNA (Fig. 3C) and ErbB2 expression was increased (21). The activity of the ErbB2 promoter construct was increased 3-fold as compared to the shRNA controls (Fig. 1A).

To further explore the mechanism of the EBP1-induced transcriptional repression, we assessed the ability of EBP1 mutants to inhibit ErbB2 promoter activity. EBP1 phosphorylation at Ser363 is required for EBP1 to bind Sin3A and inhibit the transcription of E2F1-regulated promoters (17). We examined the ability of the non-phosphorylatable S363A and phosphomimetic S363D mutants to affect ErbB2 promoter activity. Previously published data indicated that the subcellular distribution of the mutants was similar to that of wild-type EBP1 (17). The expression of wild-type and the EBP1 mutants was approximately the same (Fig. 1B, upper image). BT474 cells were transfected with pRL-TK, ErbB2-Luc and either CMV10, CMV10-EBP1, CMV10-EBP1 S363A or EBP1 S363D. CMV10-EBP1 significantly repressed ErbB2 promoter activity by 79% as compared to the vector control (P<0.001). The S363A mutation significantly reduced EBP1-mediated transcriptional repression (P<0.05). The activity of the S363D mutant was not significantly different than that of the wild-type (Fig. 1B).

Distal ErbB2 promoters are important in regulating ErbB2 levels in ErbB2-overexpressing cells (23). The ability of EBP1 to affect the activity of reporter vectors encoding -6007 and -3798 sites upsteam of the transcriptional start site were assessed. We discovered that EBP1 inhibited the activity of the -3798 promoter by 71% and the -6007 by 90% (Fig. 1C).

We used ChIP assays to determine whether EBP1 may assemble on endogenous proximal and distal ErbB2 promoters. Although the proximal ErbB2 promoter is active in cell lines expressing both high and low levels of ErbB2, distal promoter sites are important in cells in which ErbB2 is overexpressed (23) (Fig. 2A). We were additionally interested in the ability of the ErbB3 ligand HRG to affect EBP1 binding. BT474 cells were serum-starved and then treated with HRG for 20 min. EBP1 associated with both the -4600 and -500 bp elements previously demonstrated to be implicated in ErbB2 transcriptional regulation (23). The distal -4600 bp site was immunoprecipitated to a lesser extent compared to the 500 bp site. In contrast, the association of EBP1 with the GAPDH promoter was not significantly different compared to the IgG control indicating that the association of EBP1 with the ErbB2 promoter was specific. HRG treatment decreased the binding of EBP1 to both recognition sites (Fig. 2).

The decreased steady-state levels of ErbB2 mRNA observed after EBP1 overexpression may have resulted from either decreased transcription or decreased mRNA stability. A previous study demonstrated that endogenous EBP1 binds AR mRNA and that ectopic expression of EBP1 decreases the levels of AR mRNA by destabilizing AR mRNA in prostate cancer cell lines (14). We first examined the association of endogenous EBP1 with endogenous ErbB2 mRNA by RNA-IP analysis. ErbB2 mRNA was detected in the EBP1 immunoprecipitates by reverse transcription using primers for the ErbB2 coding region. As reported, HuR also bound ErbB2 mRNA (22) (Fig. 3A). Actin mRNA was not enriched in EBP1 immunoprecipitates compared to the control IgG (data not shown). To examine if EBP1 is involved in the post-transcriptional regulation of ErbB2, we measured the half-life of ErbB2 mRNA in control and BT474 cells expressing GFP-EBP1 in actinomycin D pulse chase experiments. ErbB2 mRNA stability was not altered by the overexpression of EBP1 (Fig. 3B). Similarly, ablation of EBP1 in T47D cells did not significantly affect overall ErbB2 mRNA stability (Fig. 3C).