RESUMEN
The candidate tumor suppressor BAP1 is a deubiquitinating enzyme (DUB) involved in the regulation of cell proliferation, although the molecular mechanisms governing its function remain poorly defined. BAP1 was recently shown to interact with and deubiquitinate the transcriptional regulator host cell factor 1 (HCF-1). Here we show that BAP1 assembles multiprotein complexes containing numerous transcription factors and cofactors, including HCF-1 and the transcription factor Yin Yang 1 (YY1). Through its coiled-coil motif, BAP1 directly interacts with the zinc fingers of YY1. Moreover, HCF-1 interacts with the middle region of YY1 encompassing the glycine-lysine-rich domain and is essential for the formation of a ternary complex with YY1 and BAP1 in vivo. BAP1 activates transcription in an enzymatic-activity-dependent manner and regulates the expression of a variety of genes involved in numerous cellular processes. We further show that BAP1 and HCF-1 are recruited by YY1 to the promoter of the cox7c gene, which encodes a mitochondrial protein used here as a model of BAP1-activated gene expression. Our findings (i) establish a direct link between BAP1 and the transcriptional control of genes regulating cell growth and proliferation and (ii) shed light on a novel mechanism of transcription regulation involving ubiquitin signaling.
Asunto(s)
Factor C1 de la Célula Huésped/química , Factor C1 de la Célula Huésped/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/metabolismo , Factor de Transcripción YY1/química , Factor de Transcripción YY1/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Bovinos , Línea Celular , Proliferación Celular , ADN/genética , ADN/metabolismo , Complejo IV de Transporte de Electrones/genética , Células HeLa , Factor C1 de la Célula Huésped/antagonistas & inhibidores , Factor C1 de la Célula Huésped/genética , Humanos , Técnicas In Vitro , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Complejos Multiproteicos , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Interferencia de ARN , Homología de Secuencia de Ácido Nucleico , Transducción de Señal , Activación Transcripcional , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/genética , Ubiquitinación , Factor de Transcripción YY1/antagonistas & inhibidores , Factor de Transcripción YY1/genéticaRESUMEN
We present a strategy for identifying off-target effects and hidden phenotypes of drugs by directly probing biochemical pathways that underlie therapeutic or toxic mechanisms in intact, living cells. High-content protein-fragment complementation assays (PCAs) were constructed with synthetic fragments of a mutant fluorescent protein ('Venus', EYFP or both), allowing us to measure spatial and temporal changes in protein complexes in response to drugs that activate or inhibit particular pathways. One hundred and seven different drugs from six therapeutic areas were screened against 49 different PCA reporters for ten cellular processes. This strategy reproduced known structure-function relationships and also predicted 'hidden,' potent antiproliferative activities for four drugs with novel mechanisms of action, including disruption of mitochondrial membrane potential. A simple algorithm identified a 25-assay panel that was highly predictive of antiproliferative activity, and the predictive power of this approach was confirmed with cross-validation tests. This study suggests a strategy for therapeutic discovery that identifies novel, unpredicted mechanisms of drug action and thereby enhances the productivity of drug-discovery research.