Identification of a Hormone-regulated Dynamic Nuclear Actin Network Associated with Estrogen Receptor α in Human Breast Cancer Cell Nuclei

Concetta Ambrosino,Roberta Tarallo,Angela Bamundo,Danila Cuomo,Gianluigi Franci,Giovanni Nassa,Ornella Paris,Maria Ravo,Alfonso Giovane,Nicola Zambrano,Tatiana Lepikhova,Olli A Jänne,Marc Baumann,Tuula A Nyman,Luigi Cicatiello,Alessandro Weisz

doi:10.1074/mcp.m900519-mcp200

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.

Highlights

Estrogen receptor ␣ (ER␣) 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
Characterization and Stable Expression of tandem affinity purification (TAP)-ER␣ Fusion Protein in MCF-7 Cells—To generate a suitable fusion protein for TAP [45], the human ER␣ coding sequence was cloned upstream of a TAP tag comprising the IgG binding domain of protein A and a calmodulin binding peptide separated by a peptide carrying a TEV protease cleavage site (Fig. 1A) to generate TAP-ER␣
We controlled whether tagging interfered with ER␣ activity on a responsive reporter gene for which transcription is driven by an estrogen-responsive minimal promoter and enhanced by estrogen response element (ERE) sequences (ERE-TK-luc; Fig. 1B)

Summary

Introduction

Estrogen receptor ␣ (ER␣) 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. The already mentioned ability of ligand-activated ERs to form multiple complexes with key intracellular regulatory molecules represents a well known mechanism to explain their multifaceted effects in key processes such as signal transduction and transcriptional regulation [2, 6] For this reason, we established an experimental model to identify and characterize the nuclear ER interactome of hormone-responsive human breast cancer cells. It has been recently demonstrated that ␤-actin and its binding partners are important regulators of several nuclear processes, including all transcriptional steps and chromatin remodeling [33,34,35,36], repositioning of actively transcribed genes to the periphery of nuclear territories [37], and enhancerpromoter interactions by intra- and interchromosomal looping [9] These findings, combined with the identification of the same proteins in stable ER␣ complexes within the nucleus, indicate that the nuclear ␤-actin pathway is part of the molecular machinery that allows control of genome activity and structural organization by estrogen via ER␣

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Results

Conclusion