RESUMEN
The four-subunit negative elongation factor (NELF) complex mediates RNA polymerase II (Pol II) pausing at promoter-proximal regions. Ablation of individual NELF subunits destabilizes the NELF complex and causes cell lethality, leading to the prevailing concept that NELF-mediated Pol II pausing is essential for cell proliferation. Using separation-of-function mutations, we show here that NELFB function in cell proliferation can be uncoupled from that in Pol II pausing. NELFB mutants sequestered in the cytoplasm and deprived of NELF nuclear function still support cell proliferation and part of the NELFB-dependent transcriptome. Mechanistically, cytoplasmic NELFB physically and functionally interacts with prosurvival signaling kinases, most notably phosphatidylinositol-3-kinase/AKT. Ectopic expression of membrane-tethered phosphatidylinositol-3-kinase/AKT partially bypasses the role of NELFB in cell proliferation, but not Pol II occupancy. Together, these data expand the current understanding of the physiological impact of Pol II pausing and underscore the multiplicity of the biological functions of individual NELF subunits.
Asunto(s)
Proteínas Proto-Oncogénicas c-akt , ARN Polimerasa II , Citoplasma/metabolismo , Fosfatidilinositoles , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Transcripción Genética , Animales , RatonesRESUMEN
COBRA1 (co-factor of BRCA1) is one of the four subunits of the negative elongation factor originally identified as a BRCA1-interacting protein. Here, we provide first-time evidence for the oncogenic role of COBRA1 in prostate pathogenesis. COBRA1 is aberrantly expressed in prostate tumors. It positively influences androgen receptor (AR) target gene expression and promoter activity. Depletion of COBRA1 leads to decreased cell viability, proliferation, and anchorage-independent growth in prostate cancer cell lines. Conversely, overexpression of COBRA1 significantly increases cell viability, proliferation, and anchorage-independent growth over the higher basal levels. Remarkably, AR-positive androgen dependent (LNCaP) cells overexpressing COBRA1 survive under androgen-deprivation conditions. Remarkably, treatment of prostate cancer cells with well-studied antitumorigenic agent, 2-methoxyestradiol (2-ME2), caused significant DNA methylation changes in 3255 genes including COBRA1. Furthermore, treatment of prostate cancer cells with 2-ME2 downregulates COBRA1 and inhibition of prostate tumors in TRAMP (transgenic adenocarcinomas of mouse prostate) animals with 2-ME2 was also associated with decreased COBRA1 levels. These observations implicate a novel role for COBRA1 in progression to CRPC and suggest that COBRA1 downregulation has therapeutic potential.
Asunto(s)
Proteína BRCA1/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/metabolismo , Factores de Transcripción/metabolismo , 2-Metoxiestradiol , Animales , Proteína BRCA1/genética , Proliferación Celular/genética , Proliferación Celular/fisiología , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Estradiol/análogos & derivados , Estradiol/farmacología , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Masculino , Ratones , Proteínas Nucleares/genética , Neoplasias de la Próstata/genética , Unión Proteica , Proteínas de Unión al ARN , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Factores de Transcripción/genéticaRESUMEN
NELF-B is a BRCA1-interacting protein and subunit (with NELF-A, -C/D, and -E) of the human negative elongation factor (NELF) complex, which participates in RNA polymerase II pausing shortly after transcription initiation, especially for synchronized gene expression. We now report new activities of NELF-B and other NELF complex subunits, which are to attenuate glucocorticoid receptor (GR)-mediated gene induction, reduce the partial agonist activity of an antagonist, and increase the EC50 of an agonist during nonsynchronized expression of exogenous and endogenous reporters. Stable knockdown of endogenous NELF-B has the opposite effects on an exogenous gene. The GR ligand-binding domain suffices for these biological responses. ChIP assays reveal that NELF-B diminishes GR recruitment to promoter regions of two endogenous genes. Using a new competition assay, NELF-A and NELF-B are each shown to act independently as competitive decelerators at two steps after the site of GR action and before or at the site of reporter gene activity. A common motif in each NELF was identified that is required for full activity of both NELF-A and NELF-B. These studies allow us to position the actions of two new modulators of GR-regulated transactivation, NELF-A and NELF-B, relative to other factors in the overall gene induction sequence.