Identification of a hormone-regulated dynamic nuclear actin network associated with estrogen receptor alpha in human breast cancer cell nuclei

Abstract

Estrogen receptor alpha (ERalpha) is a modular protein of the steroid/nuclear receptor family of transcriptional regulators that upon binding to the hormone undergoes structural changes, resulting in its nuclear translocation and docking to specific chromatin sites. In the nucleus, ERalpha assembles in multiprotein complexes that act as final effectors of estrogen signaling to the genome through chromatin remodeling and epigenetic modifications, leading to dynamic and coordinated regulation of hormone-responsive genes. Identification of the molecular partners of ERalpha and understanding their combinatory interactions within functional complexes is a prerequisite to define the molecular basis of estrogen control of cell functions. To this end, affinity purification was applied to map and characterize the ERalpha interactome in hormone-responsive human breast cancer cell nuclei. MCF-7 cell clones expressing human ERalpha fused to a tandem affinity purification tag were generated and used to purify native nuclear ER-containing complexes by IgG-Sepharose affinity chromatography and glycerol gradient centrifugation. Purified complexes were analyzed by two-dimensional DIGE and mass spectrometry, leading to the identification of a ligand-dependent multiprotein complex comprising beta-actin, myosins, and several proteins involved in actin filament organization and dynamics and/or known to participate in actin-mediated regulation of gene transcription, chromatin dynamics, and ribosome biogenesis. Time course analyses indicated that complexes containing ERalpha and actin are assembled in the nucleus early after receptor activation by ligands, and gene knockdown experiments showed that gelsolin and the nuclear isoform of myosin 1c are key determinants for assembly and/or stability of these complexes. Based on these results, we propose that the actin network plays a role in nuclear ERalpha actions in breast cancer cells, including coordinated regulation of target gene activity, spatial and functional reorganization of chromatin, and ribosome biogenesis.

Fig. 1.

Functional analysis and stable expression in MCF-7 cells of TAP-tagged ERα. A, schematic representation of the ERα-TAP tag fusion protein generated for this study. hERα, human ERα coding sequence (595 aa); CBP, calmodulin binding peptide (29 aa); TEV, peptide comprising a TEV protease cleavage site (18 aa); Pr-A, Staphylococcus aureus protein A (137 aa). B, the transcriptional activity of wt ERα and the TAP-ERα fusion protein was assayed by transient transfection in MDA-MB-231 cells. The cells, E₂-deprived for 5 days, were co-transfected with the expression vectors for ERα and TAP-ERα or the respective control vectors (pSG5 and pUseAmp⁺C-TAP) and the reporter plasmid ERE-TK-luc; 24 h post-transfection cells were either treated with vehicle alone (control) or stimulated with E₂ for 24 h, and luciferase activity was assayed in whole-cell extracts. Results (±S.E.) are representative of two experiments performed in triplicate. C, MCF-7 cells were stably transfected with expression vectors for the TAP tag alone (lanes 1) or for TAP-ERα (lanes 2). The expression of the tagged receptor and its ratio to the endogenous receptor were assayed by WB of protein extracts from selected clones. Results shown refer to the cell clone used for the further experiments. D, analysis of cell cycle progression after estrogen stimulation of MCF-7-derived cell clones expressing the TAP tag alone or TAP-ERα. The percentage of S + G₂ phase cells was determined by flow cytometry in estrogen-starved cultures 27 h after treatment with vehicle alone (EtOH) or the indicated concentrations of E₂. Results (±S.E.) are representative of three experiments performed in triplicate.

Fig. 2.

TAP-ERα protein complex purification. A, schematic representation of the first steps of the tandem affinity purification procedure applied for isolation of native ERα-containing complexes from MCF-7 cell nuclei. B and C, nuclear extracts from TAP- (B) and TAP-ERα (C)-expressing cells were isolated and purified as described above, and exogenous protein recovery was monitored by WB using a specific Ab against human ERα. In both cases, the relative concentrations of both forms of the receptor are shown before and after addition of IgG-Sepharose (lanes 1 and 2), bound to IgG-Sepharose before and after TEV cleavage (lanes 3 and 4), and in eluates obtained by two sequential TEV treatments (lanes 5 and 6). To avoid saturation of the signal, different amounts of samples were loaded in each case as follows: 1:400 for nuclear extract (lanes 1 and 2), 1:200 for IgG bead slurry (lanes 3 and 4), and 1:100 for each TEV eluate (lanes 5 and 6). *ns, nonspecific band detected by the Abs used; PROT A, protein A; CBP, calmodulin binding peptide.

Fig. 3.

DIGE two-dimensional gel of proteins after tandem affinity purification. TEV eluates were obtained by purification of nuclear proteins (50 mg) from TAP- and TAP-ERα-expressing cells; they were labeled with Cy2 and Cy3 dye, respectively, before separation by two-dimensional gel electrophoresis. The TAP-ERα-specific spots, circled in the image of the SYPRO staining (lower panel), were excised and analyzed by LC-MS/MS. The identified proteins are listed in Table I. Arrows and numbers refer to the position and molecular mass of the four proteins (A–D) identified by MS analysis.

Fig. 4.

17β-Estradiol promotes time-dependent recruitment of β-actin and actin-interacting proteins to TAP-ERα in MCF-7 cell nuclei. A, TAP-ERα-expressing cells were E₂-deprived (0) and subsequently stimulated for the indicated times (′, minutes), and 1 mg of nuclear proteins was incubated in each case with IgG-Sepharose and, upon binding and extensive washing, directly analyzed by WB, probing the membrane with specific Abs for the indicated proteins. The results shown in the figure were obtained on the same membrane and are representative of replicate experiments. B, cytoplasmic extracts (50 mg) prepared from TAP-ERα-expressing cells stimulated with E₂ for 2 h were prepared as described under “Experimental Procedures” and processed as reported in Fig. 2A. To avoid saturation of the signal, different amounts of samples were loaded in each case as follows: 1:400 for cytosolic extract (lanes 1 and 2), 1:40 for IgG bead slurry (lanes 3 and 4), and 1:25 for each TEV eluate (lanes 5 and 6). *ns, nonspecific band detected by the Abs used.

Fig. 5.

Hormone-dependent association of ERα, β-actin, and actin-interacting proteins in MCF-7 cell nuclei. A, hormone-starved MCF-7 cells were transfected with an expression vector encoding FLAG-β-actin and treated either with EtOH (negative control) or 10⁻⁸ m E₂ for 1 h. Nuclear extracts (0.5 mg of proteins) were immunoprecipitated (IP) with anti-FLAG Abs and analyzed by WB with either anti-FLAG or anti-ERα Abs. Samples analyzed are as follows: nuclear extracts before (lanes 1 and 5) and after (lanes 2 and 6) incubation with the affinity resin and bound (lanes 3 and 7) and eluted (lanes 4 and 8) proteins. B, MCF-7 nuclear extracts (0.5 mg of protein; lane 1) were immunoprecipitated with anti-ERα Abs (lane 2) or unrelated antibodies (anti-green fluorescent protein; lane 3) and analyzed by WB, probing the membrane with specific Abs for the indicated proteins. The results obtained on the same membrane are shown in the figure and are representative of results obtained in multiple experiments. C, hormone-starved MCF-7 cells were transfected with the expression vectors for either FLAG-β-actin, FLAG-ERα, or FLAG alone (p3XFLAG-CMV) and stimulated with 10⁻⁸ m E₂ for 1 h. Nuclear extracts (lanes 1, 3, and 5) were immunoprecipitated with anti-FLAG Abs, and the immunoprecipitates were analyzed by WB for the presence of the indicated proteins (lanes 2, 4, and 6). The data shown are representative of two experiments and were obtained by hybridization of the same membrane with the indicated Abs.

Fig. 6.

In vivo co-localization of ERα and β-actin on the E₂-responsive pS2/TFF1 gene promoter following estrogen stimulation, induction of ERα-β-actin complex formation by tamoxifen and effects of cytochalasin D on ER-mediated gene activation. A, ChIP experiments were performed on chromatin prepared from MCF-7 cells deprived of hormone and stimulated with EtOH or 10⁻⁸ m E₂ for 45 min before in vivo chromatin cross-linking, extraction, immunoprecipitation, and analysis as described under “Experimental Procedures.” no ab, negative control. The results refer to replicate experiments carried out in triplicate (±S.E.). B, nuclear extracts were prepared from TAP-ERα-expressing cells that were hormone-starved and then treated with 10⁻⁸ m E₂ or tamoxifen (TAM) for 45 min. 7 mg of proteins were subjected to IgG-Sepharose binding and TEV elution before WB with specific Abs against the indicated proteins. IgG-bound samples (lanes 1 and 4), IgG-bound samples after TEV cleavage (lanes 2 and 5), TEV-eluted samples (lanes 3 and 6) are shown. C, MELN, MCF-7 cells carrying the luciferase gene under the control of a minimal E₂-responsive promoter stably integrated in the genome, were hormone-starved for 5 days before treatment for the indicated times with EtOH, E₂, or tamoxifen. Where indicated, Cyt D (5 μm) was added to the cultures either 3 h before (3-h time point) or together with EtOH or ER ligands (6-h time point), and cells were harvested at the indicated times for analysis of luciferase activity. Luciferase activity values reported were normalized to the protein content of each extract. Results (±S.E.) are representative of two experiments performed in triplicate. CBP, calmodulin binding peptide; RLU, relative light units.

Fig. 7.

Association of hormone-activated ERα to F-actin and evidence for high molecular weight ER-containing complexes in crude and affinity-purified nuclear extracts from hormone-stimulated MCF-7 cells. A, association of ERα to F-actin was assayed in MCF-7 cells under normal growing conditions. Nuclei from actively growing cells were prepared as reported under “Experimental Procedures” and processed to separate G- and F-actin. The separated fractions were analyzed by WB. B and C, nuclear extracts (2 mg of proteins; B) or IgG-Sepharose-purified samples (40 μg of TEV eluate proteins; C) from hormone-starved TAP-ERα cells stimulated with E₂ (10⁻⁸ m) for 2 h were fractionated on 5–45% glycerol gradients as described under “Experimental Procedures.” 0.5-ml fractions were collected from the top of each gradient, and 30-μl aliquots of the indicated fractions were analyzed by WB. The data shown in both panels are representative of two experiments and were obtained by hybridization of the same membrane with the indicated Abs with the exception of data relative to myosin 1c in B. ns*, nonspecific band detected by anti-myosin 1c Abs; CBP, calmodulin binding peptide.

Fig. 8.

Lentivirus-mediated gene knockdown analysis of role of gelsolin, flightless I, and myosin 1c in ERα-β-actin complex formation. MCF-7 cells were infected with lentiviruses expressing shRNA for gelsolin (GSN₍₂₎), flightless I (FLII₍₃₎), and myosin 1c (MYO₍₂₎). Nuclear extracts (1 mg) from control (Ctrl) or knockdown cells maintained under normal growing conditions were immunoprecipitated (IP) with an anti-ERα Ab. Nuclear extracts (lanes 1, 3, 5, and 7) and immunoprecipitated samples (lanes 2, 4, 6, and 8) were analyzed by WB, probing the membrane with specific Abs for the indicated proteins. Results obtained on the same membrane are shown in the figure and are representative of two independent infections and, in each case, of replicate analyses. *ns, nonspecific band detected by the Abs used; Pol, RNA polymerase.

Fig. 9.

Identification of ERα-β-actin protein network in hormone-stimulated MCF-7 cell nuclei. To draw the map of ERα and β-actin interactions, the list of proteins identified by mass spectrometry analysis of IgG-Sepharose-purified TAP-ERα samples was matched to the list of known β-actin-interacting proteins present in the UniHI database. Solid lines connecting proteins mark direct protein-protein interactions. FLII is reported in gray because it was identified in this study by WB but not by MS analysis; the dotted line and the arrow recapitulate the experimental results described here.