The role of retinoblastoma-associated proteins 46 and 48 in estrogen receptor alpha mediated gene expression

Abstract

The differential recruitment of coregulatory proteins to the DNA-bound estrogen receptor alpha (ERalpha) plays a critical role in mediating estrogen-responsive gene expression. We previously isolated and identified retinoblastoma-associated proteins 46 (RbAp46) and 48 (RbAp48), which are associated with chromatin remodeling, histone deacetylation, and transcription repression, as proteins associated with the DNA-bound ERalpha. We now demonstrate that RbAp46 and RbAp48 interact with ERalphain vitro and in vivo, associate with ERalpha at endogenous, estrogen-responsive genes, and alter expression of endogenous, ERalpha-activated and -repressed genes in MCF-7 breast cancer cells. Our findings reveal that RbAp48 limits expression of estrogen-responsive genes and that RbAp46 modulates estrogen responsiveness in a gene-specific manner. The ability of RbAp46 and RbAp48 to interact with ERalpha and influence its activity reveals yet another role for these multifunctional proteins in regulating gene expression.

Fig. 1. Expression of RbAp46 and RbAp48

Whole cell extracts were prepared from MCF-7 breast cancer cells that had been treated with ethanol or 10 nM E₂ for the indicated times and subjected to Western analysis with antibody directed against RbAp46 and RbAp48, ERα or GAPDH. The blot shown is representative of six independent experiments.

Fig. 2. Localization of RbAp46, RbAp48, and ERα in MCF-7 cells

MCF-7 cells, which were maintained in a hormone-free environment, were fixed with formaldehyde and incubated with an antibody directed against ERα, RbAp46 (A) or RbAp48 (B). ERα (green) and RbAp proteins (red) are shown individually and as merged ERα, RbAp images (yellow). Control cells, which had not been exposed to primary antibody, are included as inserts. DAPI was used to visualize MCF-7 nuclei. Results are representative of multiple fields in three independent experiments.

Fig. 3. Interaction of RbAp46 and RbAp48 with ERα

(A) Bacterially expressed GST, GST-tagged RbAp46, or GST-tagged RbAp48 was immobilized on glutathione beads and incubated with nuclear extracts from MCF-7 cells that had been treated with ethanol or E₂ for 20 min. Eluted proteins were subjected to Western analysis with an ERα specific antibody. Input (10%) was included (lanes 1 and 2) to demonstrate that equivalent amounts of ERα, RbAp46, and RbAp48 were present in the extracts from ethanol and E2 treated cells. (B) Extracts from MCF-7 cells that had been treated with ethanol or 10 nM E₂ for 20 min were incubated with IgG, an RbAp46-specific, or an RbAp48-specific antibody. Specifically-bound proteins were eluted and subjected to Western analysis using an ERα-specific antibody (top panel) or antibodies directed against RbAp46 and RbAp48 (bottom panel). Input (2%) was included (lanes 1 and 2) to demonstrate that equivalent amounts of ERα, RbAp46, and RbAp48 were present in the extracts from ethanol and E2 treated cells. Data from 3 independent experiments were combined and are presented as the mean ± SEM to the right of each blot.

Fig. 4. Effect of RbAp46 on endogenous, estrogen-responsive genes

MCF-7 breast cancer cells were transfected with control or RbAp46-specific siRNA and then treated with ethanol (gray bars) or 10 nM E₂ (black bars) for 24 hrs. Whole cell extracts (30 µg) were subjected to Western analysis using an antibody directed against both RbAp46 and RbAp48 or GAPDH. The levels of mRNA were quantified in triplicate using real time PCR with gene specific primers. ANOVA analysis was performed using SAS 9.1. An asterisk indicates a significant difference (p<0.05) in the E₂-treated cells compared to ethanol-treated cells and a plus sign indicates a significant difference (p<0.05) in the RbAp46-specific siRNA compared to the corresponding control siRNA. Error bars are sometimes too small to be visualized. Data is representative of three independent experiments.

Fig. 5. Effect of RbAp48 on endogenous, estrogen-responsive genes

MCF-7 breast cancer cells were transfected with control or RbAp48-specific siRNA and then treated with ethanol (gray bars) or 10 nM E₂ (black bars) for 24 hrs. Whole cell extracts (30 µg) were subjected to Western analysis using an antibody directed against both RbAp46 and RbAp48 or GAPDH. The levels of mRNA were quantified in triplicate using real time PCR with gene specific primers. ANOVA analysis was performed using SAS 9.1. An asterisk indicates a significant difference (p<0.05) in the E₂-treated cells compared to ethanol-treated cells and a plus sign indicates a significant difference (p<0.05) in the RbAp48-specific siRNA compared to the corresponding control siRNA. Error bars are sometimes too small to be visualized. Data is representative of three independent experiments.

Fig. 6. Association of ERα, RbAp46, and RbAp48 with endogenous, estrogen-responsive genes

MCF-7 cells were treated with ethanol vehicle (gray bars) or E₂ for 45 min (hatched bars) or 4 hrs (black bars) and subjected to ChIP analysis. Chromatin was immunoprecipitated with antibody directed against fluorescein (FL), ERα, RbAp46, or RbAp48. Quantitative real time PCR was performed in triplicate with primers specific to the Sox9 (A) or pS2 (B) gene. Standard curves were utilized to determine the number of genomic copies immunoprecipitated. Data shown is representative of five independent experiments and is the mean ± S.D. ANOVA analysis was performed using SAS 9.1. An asterisk indicates a significant difference (p<0.05) in the E₂-treated cells compared to ethanol treated cells. Error bars are sometimes too small to be visualized.