Identification of a Hormone-regulated Dynamic Nuclear Actin Network Associated with Estrogen Receptor α in Human Breast Cancer Cell Nuclei*
| Autor: | Luigi Cicatiello, Danila Cuomo, Gianluigi Franci, Angela Bamundo, Roberta Tarallo, Concetta Ambrosino, Olli A. Jänne, Giovanni Nassa, Nicola Zambrano, Ornella Paris, Alfonso Giovane, Tatiana Lepikhova, Marc Baumann, Tuula A. Nyman, Alessandro Weisz, Maria Ravo |
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| Jazyk: | angličtina |
| Rok vydání: | 2010 |
| Předmět: |
Time Factors
Recombinant Fusion Proteins Actin filament organization Estrogen receptor Breast Neoplasms macromolecular substances Biology Biochemistry Interactome Models Biological Chromatin remodeling Chromatography Affinity Mass Spectrometry Analytical Chemistry 03 medical and health sciences 0302 clinical medicine Cell Line Tumor Humans Electrophoresis Gel Two-Dimensional Molecular Biology Estrogen receptor beta 030304 developmental biology Cell Nucleus 0303 health sciences Estradiol Research Lentivirus Estrogen Receptor alpha Molecular biology Actins Cell biology Chromatin Neoplasm Proteins Nuclear receptor 030220 oncology & carcinogenesis Gene Knockdown Techniques Multiprotein Complexes Female Estrogen receptor alpha Chromatography Liquid |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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