RESUMEN
Members of the bromodomain and extra-terminal domain (BET) family of proteins (bromodomain-containing (BRD) 2, 3, 4, and T) are widely expressed and highly conserved regulators of gene expression in eukaryotes. These proteins have been intimately linked to human disease, and more than a dozen clinical trials are currently underway to test BET-protein inhibitors as modulators of cancer. However, although it is clear that these proteins use their bromodomains to bind both histones and transcription factors bearing acetylated lysine residues, the molecular mechanisms by which BET family proteins regulate gene expression are not well defined. In particular, the functions of the other domains such as the ET domain have been less extensively studied. Here, we examine the properties of the ET domain of BRD3 as a protein/protein interaction module. Using a combination of pulldown and biophysical assays, we demonstrate that BRD3 binds to a range of chromatin-remodeling complexes, including the NuRD, BAF, and INO80 complexes, via a short linear "KIKL" motif in one of the complex subunits. NMR-based structural analysis revealed that, surprisingly, this mode of interaction is shared by the AF9 and ENL transcriptional coregulators that contain an acetyl-lysine-binding YEATS domain and regulate transcriptional elongation. This observation establishes a functional commonality between these two families of cancer-related transcriptional regulators. In summary, our data provide insight into the mechanisms by which BET family proteins might link chromatin acetylation to transcriptional outcomes and uncover an unexpected functional similarity between BET and YEATS family proteins.
Asunto(s)
Ensamble y Desensamble de Cromatina , Péptidos/química , Proteínas de Unión al ARN/metabolismo , Transactivadores/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Acetilación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Fenómenos Biofísicos , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/fisiología , Redes Reguladoras de Genes , Células HEK293 , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , Dominios Proteicos , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/fisiología , Homología de Secuencia de Aminoácido , Transactivadores/química , Factores de TranscripciónRESUMEN
The nucleosome remodelling and deacetylase (NuRD) complex is essential for the development of complex animals. NuRD has roles in regulating gene expression and repairing damaged DNA. The complex comprises at least six proteins with two or more paralogues of each protein routinely identified when the complex is purified from cell extracts. To understand the structure and function of NuRD, a map of direct subunit interactions is needed. Dozens of published studies have attempted to define direct inter-subunit connectivities. We propose that conclusions reported in many such studies are in fact ambiguous for one of several reasons. First, the expression of many NuRD subunits in bacteria is unlikely to lead to folded, active protein. Second, interaction studies carried out in cells that contain endogenous NuRD complex can lead to false positives through bridging of target proteins by endogenous components. Combining existing information on NuRD structure with a protocol designed to minimize false positives, we report a conservative and robust interaction map for the NuRD complex. We also suggest a 3D model of the complex that brings together the existing data on the complex. The issues and strategies discussed herein are also applicable to the analysis of a wide range of multi-subunit complexes. ENZYMES: Micrococcal nuclease (MNase), EC 3.1.31.1; histone deacetylase (HDAC), EC 3.5.1.98.