ABSTRACT
The proto-oncoprotein SYT is involved in the unique translocation t(X;18) found in synovial sarcoma SYT-SSX fusions. SYT has a conserved N-terminal domain (SNH domain) that interacts with the human paralog of Drosophila Brahma (hBRM) and Brahma-related gene 1 (BRG1) chromatin remodeling proteins and a C-terminal transactivating sequence rich in glutamine, proline, glycine, and tyrosine (QPGY domain). Here we reported the isolation of the ribonucleoprotein SYT-interacting protein/co-activator activator (SIP/CoAA), which specifically binds the QPGY domain of SYT and also the SYT-SSX2 translocation fusion. SIP/CoAA is a general nuclear co-activator and an RNA splicing modulator that contains two RNA recognition motifs and multiple hexapeptide repeats. We showed that the region consisting of the hexapeptide motif (YQ domain) is similar to the hexapeptide repeat domain found in EWS and in TLS/FUS family proteins. The YQ domain also resembles the QPGY region of SYT itself and like all these other domains acts as a transcriptional activator in reporter assays. Most interestingly, the last 84 amino acids adjacent to YQ down-modulate by 25-fold the YQ transactivation of the reporter gene, and both domains are important for SIP/CoAA binding to SYT. In addition, SYT acts together with SIP/CoAA in stimulating estrogen and glucocorticoid receptor-dependent transcriptional activation. Activation is hormone-dependent and requires functional hBRM and/or BRG1. The stimulation is strongly reduced if the N-terminal region of hBRM/BRG1 (amino acids 1-211) is deleted. This region encompasses the SNF11 binding domain (amino acids 156-211), which interacts specifically with SYT in vivo and in vitro.
Subject(s)
Cell Cycle Proteins/chemistry , Cell Nucleus/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Neoplasm Proteins/chemistry , Proto-Oncogene Proteins/metabolism , RNA-Binding Protein EWS/chemistry , RNA-Binding Protein FUS/chemistry , RNA-Binding Proteins/chemistry , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , COS Cells , Cell Line , Cell Line, Tumor , Chlorocebus aethiops , Chromatin/chemistry , Cloning, Molecular , Cytoplasm/metabolism , DNA, Complementary/metabolism , Down-Regulation , Drosophila , Gene Library , Glutamine/chemistry , Glutathione Transferase/metabolism , Glycine/chemistry , Hormones/metabolism , Humans , Immunoblotting , Immunoprecipitation , Intracellular Signaling Peptides and Proteins/metabolism , Ligands , Models, Biological , Molecular Sequence Data , Plasmids/metabolism , Proline/chemistry , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , RNA Splicing , Recombinant Fusion Proteins/chemistry , Sarcoma, Synovial/metabolism , Sequence Homology, Amino Acid , Serine-Arginine Splicing Factors , Transcription, Genetic , Transcriptional Activation , Transfection , Translocation, Genetic , Two-Hybrid System Techniques , Tyrosine/chemistryABSTRACT
The BAH domain (bromo-associated homology domain) was first identified from a repeated motif found in the nuclear protein polybromo--a large (187 kDa) modular protein comprising six bromodomains, two BAH domains and an HMG box. To date, the BAH domain has no ascribed function, although it is found in a wide range of proteins that contain additional domains involved in either transcriptional regulation (e.g. SET, PHD and bromodomain) and/or DNA binding (HMG box and AT hook). The molecular function of polybromo itself also remains unclear, but it has been identified as a key component of an SWI/SNF (switching/sucrose non-fermenting)-related, ATP-dependent chromatin-remodelling complex PBAF (polybromo, BRG1-associated factors; also known as SWI/SNF-B or SWI/SNFbeta). We present in this paper the crystal structure of the proximal BAH domain from chicken polybromo (BAH1), at a resolution of 1.6 A (1 A=0.1 nm). Structure-based sequence analysis reveals several features that may be involved in mediating protein-protein interactions.
Subject(s)
Nuclear Proteins/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Animals , Chickens , Conserved Sequence , Crystallization , DNA-Binding Proteins , Molecular Sequence Data , Protein Structure, Tertiary , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Many studies have now established that the SWI/SNF chromatin remodelling complexes are involved in activation and repression of a variety of genes. In mammalian cells, these complexes contain the BRM and BRG1 helicase-like proteins that are thought to be responsible for nucleosome remodelling. The proto-oncoprotein SYT, involved in the unique translocation t(X;18) found in synovial sarcoma, is known to interact with human BRM (hBRM), thus providing a link between chromatin remodelling factors and human cancer. In this work, we address how SYT interacts with hBRM and BRG1. We demonstrate that the conserved N-terminal SNH domain of SYT, which is also present in the oncoproteins SYT-SSX, binds to both hBRM and BRG1. We have also found that in vivo the C-terminus transactivation QPGY region of SYT can interact with itself. This results in an amplified interaction with hBRM and highlights a possible regulatory function of this domain in cells.