ANCCA, an estrogen-regulated AAA+ ATPase coactivator for ERalpha, is required for coregulator occupancy and chromatin modification

Abstract

AAA+ proteins play crucial roles in diverse biological processes via their ATPase-driven remodeling of macromolecular complexes. Here we report our identification of an evolutionarily conserved AAA+ protein, ANCCA/pro2000, endowed with a bromodomain that is strongly induced by estrogen in human breast cancer cells and is a direct target of protooncogene ACTR/AIB1/SRC-3. We found that ANCCA associates directly with estrogen-bound estrogen receptor (ER) alpha and ACTR. It is selectively recruited, upon estrogen stimulation, to a subset of ERalpha target genes including cyclin D1, c-myc, and E2F1 and is required for their estrogen-induced expression as well as breast cancer cell proliferation. Further studies indicate that ANCCA binds and hydrolyzes ATP and is critical for recruitment of coregulator CBP and histone hyperacetylation at the ER target chromatin. Moreover, mutations at the ATP binding motifs rendered ANCCA defective as a coactivator in mediating estrogen induction of gene expression. Together, our findings reveal an unexpected layer of regulatory mechanism in hormone signaling mediated by ANCCA and suggest that hormone-induced assembly of transcriptional coregulator complexes at chromatin is a process facilitated by AAA+ ATPase proteins.

Fig. 1.

ANCCA is a direct target of ACTR and is strongly induced by E2. (A) Schematics of ANCCA subdomains. (B) ANCCA expression in T-47D cells was analyzed by real-time RT-PCR with cells treated with anti-E2 ICI182,780 (10⁻⁸ M) for 36 h, then infected with adenovirus vectors for GFP or ACTR (lanes 1 and 2) for 24 h or with cells treated with vehicle or 10⁻⁸ M E2 (lanes 3 and 4). (C) Northern blot analysis of ANCCA expression in T-47D cells treated with E2 and MCF-10A cells in regular growth medium. (D) Western blot analysis of ANCCA expression in cells treated with E2 for 12 or 24 h.

Fig. 2.

ANCCA knockdown inhibits E2-stimulated cell proliferation and G₁–S progression. (A) T-47D cells being deprived of hormone were infected with equal titers of adeno-shRNA specifically targeting GFP (Gi) or ANCCA (Ai) and harvested at different days after E2 treatment for Western blot analysis. (B) T-47D cells being deprived of hormone were infected with the adeno-shRNAs as in A and 2 days later treated with 10⁻⁸ M E2 (+) or vehicle (−) for different days before being harvested for cell numeration.

Fig. 3.

ANCCA depletion inhibits selectively E2-stimulated expression of cyclin D1, c-myc, and E2F1. T-47D cells deprived of hormone were infected with adeno-shRNA targeting GFP (Gi) or ANCCA (Ai), treated with E2 for the indicated hours, and harvested for quantitative mRNA analysis (A) or harvested at different days after E2 treatment for Western blot analysis (B). Fold induction was obtained by comparing real-time RT-PCR data from cells not treated with E2 and cells treated for 6 or 12 h.

Fig. 4.

ANCCA is selectively recruited to a subset of ERα target genes, and its knockdown suppresses E2-induced coregulator recruitment and histone hyperacetylation. (A) Hormone-deprived MCF-7 cells were either untreated or treated with E2 for 1 or 6 h before being harvested for ChIP assay with indicated antibodies. Precipitated DNA was analyzed for protein occupancy at indicated promoters by semiquantitative PCR. The entire experiment was repeated three times. (B–D) MCF-7 cells were transfected with either ANCCA siRNA or control siRNA, maintained in hormone-depleted medium for 3 days, and then treated with E2 for 1 h before being harvested for ChIP assays with the indicated antibodies. Relative occupancy was determined by quantifying ChIP PCR products obtained from three experiments, with data from cells transfected with control siRNA and without E2 treatment set at 1. Each bar represents the average from three experiments. (E) E2-induced CBP occupancy at promoters indicated at the bottom was analyzed by ChIP assay as in A–C. No significant difference was observed between non-E2-treated cells that were transfected with either control siRNA or ANCCA siRNA.

Fig. 5.

ANCCA directly associates with ERα and ACTR. (A) Endogenous ANCCA, ERα, and ACTR protein complexes were analyzed by coimmunoprecipitation assay. Nuclear extracts from MCF-7 cells treated with E2 for 45 min were immunoprecipitated with anti-ANCCA antibody or normal rabbit IgG and blotted by indicated antibodies at the left. Input represents 15% of the extract used in the coimmunoprecipitation. (B) Direct interactions between ANCCA and ERα or ACTR were analyzed by GST pull-down assay using purified proteins. Flag-ANCCA protein (≈100 ng) was incubated with GST-H-Ras (as negative control), GST-ERα, GST-RARβ, or GST-ACTR immobilized on glutathione beads in the presence or absence of E2 (10⁻⁷ M) or all-trans retinoic acid (10⁻⁷ M). ANCCA retained on the beads was detected by Western blotting. GST fusions were stained with Ponceau S. Input represents 20% of ANCCA proteins used in the individual pull-down.

Fig. 6.

ANCCA ATPase activity is critical for its function as an ERα coactivator. (A) ANCCA binds ATP. Purified, recombinant ANCCA, or control proteins BSA and purified androgen receptor (rAR), and [γ-³²P]ATP were cross-linked by UV irradiation. Treated mixtures were separated by SDS/PAGE. Gels were stained with Coomassie blue or exposed to x-ray film. (B) ANCCA hydrolyzes ATP. Purified, recombinant ANCCA or control ACTR protein was analyzed for ATPase activity as described in Materials and Methods. ATP and its product, ADP, were separated by thin-layer chromatography and imaged by PhosphorImager. (C) A luciferase reporter assay was performed in CV-1 cells with 3× ERE-luc and expression constructs indicated for ERα and ANCCA (wild type or mutant M-AAA-1 with Walker A and Walker motifs mutated in AAA-D1 module). Transfected cells were treated with 10⁻⁹ M E2 for 36 h before harvesting for β-gal and luciferase assays. (D) Hormone-deprived cells were infected with adeno-vectors for GFP, wild type, or the mutant ANCCA. Two days after infection, cells were treated with 10⁻¹⁰ M E2 for 12 h before being harvested for real-time RT-PCR analysis or for Western blotting. Fold increase in cyclin D1 and E2F1 mRNA levels was obtained by comparing the quantitative PCR data from E2-treated cells infected with adeno-GFP with the data from E2-treated cells infected with adeno-ANCCA.