RESUMO
High-throughput (HT) in vitro methods for measuring protein-DNA binding have become invaluable for characterizing transcription factor (TF) complexes and modeling gene regulation. However, current methods do not utilize endogenous proteins and, therefore, do not quantify the impact of cell-specific post-translational modifications (PTMs) and cooperative cofactors. We introduce the HT nextPBM (nuclear extract protein-binding microarray) approach to study DNA binding of native cellular TFs that accounts for PTMs and cell-specific cofactors. We integrate immune-depletion and phosphatase treatment steps into our nextPBM pipeline to characterize the impact of cofactors and phosphorylation on TF binding. We analyze binding of PU.1/SPI1 and IRF8 from human monocytes, delineate DNA-sequence determinants for their cooperativity, and show how PU.1 affinity correlates with enhancer status and the presence of cooperative and collaborative cofactors. We describe how nextPBMs, and our accompanying computational framework, can be used to discover cell-specific cofactors, screen for synthetic cooperative DNA elements, and characterize TF cooperativity.
Assuntos
Núcleo Celular/química , Redes Reguladoras de Genes , Análise Serial de Proteínas/métodos , Fatores de Transcrição/análise , Fatores de Transcrição/metabolismo , Extratos Celulares/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Proteínas Nucleares/análise , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genética , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Células THP-1RESUMO
Transcription factors IRF3, IRF5 and IRF7 (IRF3/5/7) have overlapping, yet distinct, roles in the mammalian response to pathogens. To examine the role that DNA-binding specificity plays in delineating IRF3/5/7-specific gene regulation we used protein-binding microarrays (PBMs) to characterize the DNA binding of IRF3/5/7 homodimers. We identified both common and dimer-specific DNA binding sites, and show that DNA-binding differences can translate into dimer-specific gene regulation. Central to the antiviral response, IRF3/5/7 regulate type I interferon (IFN) genes. We show that IRF3 and IRF7 bind to many interferon-stimulated response element (ISRE)-type sites in the virus-response elements (VREs) of IFN promoters. However, strikingly, IRF5 does not bind the VREs, suggesting evolutionary selection against IRF5 homodimer binding. Mutational analysis reveals a critical specificity-determining residue that inhibits IRF5 binding to the ISRE-variants present in the IFN gene promoters. Integrating PBM and reporter gene data we find that both DNA-binding affinity and affinity-independent mechanisms determine the function of DNA-bound IRF dimers, suggesting that DNA-based allostery plays a role in IRF binding site function. Our results provide new insights into the role and limitations of DNA-binding affinity in delineating IRF3/5/7-specific gene expression.
Assuntos
Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Fatores Reguladores de Interferon/metabolismo , Elementos de Resposta , Sítios de Ligação , DNA/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/química , Fator Regulador 7 de Interferon/química , Fatores Reguladores de Interferon/química , Interferon Tipo I/genética , Análise Serial de Proteínas , Multimerização ProteicaRESUMO
Protein-DNA binding is central to specificity in gene regulation, and methods for characterizing transcription factor (TF)-DNA binding remain crucial to studies of regulatory specificity. High-throughput (HT) technologies have revolutionized our ability to characterize protein-DNA binding by significantly increasing the number of binding measurements that can be performed. Protein-binding microarrays (PBMs) are a robust and powerful HT platform for studying DNA-binding specificity of TFs. Analysis of PBM-determined DNA-binding profiles has provided new insight into the scope and mechanisms of TF binding diversity. In this review, we focus specifically on the PBM technique and discuss its application to the study of TF specificity, in particular, the binding diversity of TF homologs and multi-protein complexes.