Estrogen receptor alpha (ER-alpha) is a modular protein of the steroid/nuclear receptor family of transcriptional regula- tors that upon binding to the hormone undergoes struc- tural changes, resulting in its nuclear translocation and docking to specific chromatin sites. In the nucleus, ER-alpha assembles in multiprotein complexes that act as final effectors of estrogen signaling to the genome through chromatin remodeling and epigenetic modifications, lead- ing to dynamic and coordinated regulation of hormone- responsive genes. Identification of the molecular partners of ER 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 char- acterize the ER-alpha interactome in hormone-responsive hu- man breast cancer cell nuclei. MCF-7 cell clones express- ing human ER-alpha fused to a tandem affinity purification tag were generated and used to purify native nuclear ER- containing complexes by IgG-Sepharose affinity chroma- tography 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 or- ganization and dynamics and/or known to participate in actin-mediated regulation of gene transcription, chroma- tin dynamics, and ribosome biogenesis. Time course anal- yses indicated that complexes containing ER-alpha and actin are assembled in the nucleus early after receptor activa- tion 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 ER-alpha actions in breast cancer cells, including coordinated regulation of target gene activity, spatial and functional reorganization of chromatin, and ribosome biogenesis.
Identification of a hormone-regulated dynamic actin network associated with estrogen receptor alpha in human breast cancer cell nuclei.
TARALLO, ROBERTA;FRANCI G;NASSA, GIOVANNI;RAVO, MARIA;WEISZ, Alessandro
2011-01-01
Abstract
Estrogen receptor alpha (ER-alpha) is a modular protein of the steroid/nuclear receptor family of transcriptional regula- tors that upon binding to the hormone undergoes struc- tural changes, resulting in its nuclear translocation and docking to specific chromatin sites. In the nucleus, ER-alpha assembles in multiprotein complexes that act as final effectors of estrogen signaling to the genome through chromatin remodeling and epigenetic modifications, lead- ing to dynamic and coordinated regulation of hormone- responsive genes. Identification of the molecular partners of ER 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 char- acterize the ER-alpha interactome in hormone-responsive hu- man breast cancer cell nuclei. MCF-7 cell clones express- ing human ER-alpha fused to a tandem affinity purification tag were generated and used to purify native nuclear ER- containing complexes by IgG-Sepharose affinity chroma- tography 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 or- ganization and dynamics and/or known to participate in actin-mediated regulation of gene transcription, chroma- tin dynamics, and ribosome biogenesis. Time course anal- yses indicated that complexes containing ER-alpha and actin are assembled in the nucleus early after receptor activa- tion 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 ER-alpha actions in breast cancer cells, including coordinated regulation of target gene activity, spatial and functional reorganization of chromatin, and ribosome biogenesis.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
